Materials and methods for increasing the tocopherol content in seed oil

ABSTRACT

The present invention relates generally to the field of molecular biology and concerns increasing the tocopherol content of a plant relative to a control plant, comprising expressing in a plant at least one polynucleotide encoding a delta-12-desaturase, at least one polynucleotide encoding a delta-6-desaturase, at least one polynucleotide encoding a delta-6-elongase, and at least one polynucleotide encoding a delta-5-desaturase. The present invention also relates to methods for the manufacture of oil, fatty acid- or lipids-containing compositions, and to such oils and lipids as such.

This application is continuation of U.S. patent application Ser. No.15/525,768, which is a National Stage application of InternationalApplication No. PCT/EP2015/076608, filed Nov. 13, 2015, which claims thebenefit of U.S. Provisional Patent Application No. 62/079,622, filedNov. 14, 2014 and U.S. Provisional Patent Application No. 62/234,373,filed Sep. 29, 2015; all of the aforementioned applications areincorporated herein by reference in their entirety.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

The Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is“150218A_Seqlisting.txt”, which was created on Apr. 19, 2019 and is1,303,596 bytes in size. The subject matter of the Sequence Listing isincorporated herein in its entirety by reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of molecularbiology and concerns increasing the tocopherol content of a plantrelative to a control plant, comprising expressing in a plant at leastone polynucleotide encoding a delta-12-desaturase, at least onepolynucleotide encoding a delta-6-desaturase, at least onepolynucleotide encoding a delta-6-elongase, and at least onepolynucleotide encoding a delta-5-desaturase. The present invention alsorelates to methods for the manufacture of oil, fatty acid- orlipids-containing compositions, and to such oils and lipids as such.

BACKGROUND OF THE INVENTION

Fatty acids are carboxylic acids with long-chain hydrocarbon side groupsthat play a fundamental role in many biological processes. Fatty acidsare rarely found free in nature but, rather, occur in esterified form asthe major component of lipids. As such, lipids/fatty acids are sourcesof energy (e.g., beta-oxidation). In addition, lipids/fatty acids are anintegral part of cell membranes and, therefore, are indispensable forprocessing biological or biochemical information.

Very long chain polyunsaturated fatty acids (VLC-PUFAs) such asdocosahexaenoic acid (DHA, 22:6(4,7,10,13,16,19)) are essentialcomponents of cell membranes of various tissues and organelles inmammals (e.g. nerve, retina, brain and immune cells). Clinical studieshave shown that DHA is essential for the growth and development of thebrain in infants, and for maintenance of normal brain function in adults(Martinetz, M. (1992) J. Pediatr. 120:S129 S138). DHA also hassignificant effects on photoreceptor function involved in the signaltransduction process, rhodopsin activation, and rod and cone development(Giusto, N. M., et al. (2000) Prog. Lipid Res. 39:315-391). In addition,some positive effects of DHA were also found on diseases such ashypertension, arthritis, atherosclerosis, depression, thrombosis andcancers (Horrocks, L. A. and Yeo, Y. K. (1999) Pharmacol. Res.40:211-215). Therefore, an appropriate dietary supply of DHA isimportant for human health. The human body is able to converteicosapentaenoic acid (EPA, 20:5(5,8,11,14,17)) into DHA. EPA isnormally found in marine food and is abundant in oily fish from theNorth Atlantic. In addition to serving as a precursor to DHA, EPA canalso be converted into eicosanoids in the human body. The eicosanoidsproduced from EPA have anti-inflammatory and anti-platelet aggregatingproperties. A large number of beneficial health effects have been shownfor DHA or mixtures of EPA and DHA.

Vitamin E (tocopherol) is a lipid soluble antioxidant that is importantfor preventing oxidative damage in both plants and animals and is knownto have a beneficial effect in the prevention of cardiovascular disease.Vitamin E naturally occurs in vegetable oils, where it functions toprevent oxidative damage. Vegetable oils therefore represent a usefulsource of vitamin E in the human diet. Additionally, vitamin E extractedfrom vegetable oils is used as an additive in other food, healthsupplement, and cosmetic products.

Up to now it has not been possible to correlate vitamin E concentrationwith any n-3 VLC-PUFA (i.e., EPA or DHA) component of oil. Vitamin Eoccurs in plants as various forms of tocopherol, including alpha-,beta-, gamma-, and delta. A study containing 52 landraces and 15breeding lines of Brassica napus revealed a significant positivecorrelation between alpha-tocopherol and 18:1+18:2, but no correlationbetween gamma-tocopherol and any fatty acid component (Li et al. (2013)J Agric Food Chem 61:34-40). Tocopherol concentrations have not beencorrelated with the degree of unsaturation in various Brassica napusseeds with genetically altered fatty acid composition (Abidi et al(1999) J Am Oil Chem Soc 76, 463-467, and Dolde et al (1999) J Am OilChem Soc 76, 349-355).

There is thus the need to provide a reliable source for plants, inparticular seeds, comprising tocopherol in preferably highconcentrations.

SUMMARY OF THE INVENTION

The invention is thus concerned with a method for increasing thetocopherol content of a plant relative to a control plant, comprisingexpressing in a plant at least one polynucleotide encoding adelta-12-desaturase, at least one polynucleotide encoding adelta-6-desaturase, at least one polynucleotide encoding adelta-6-elongase, and at least one polynucleotide encoding adelta-5-desaturase.

In an embodiment, the method further comprises expressing in the plantat least one polynucleotide encoding an omega-3-desaturase.

In an embodiment, the method further comprises expressing in the plantat least one polynucleotide encoding a delta-5-elongase.

In an embodiment, the method further comprises expressing in the plantat least one polynucleotide encoding a delta-4-desaturase. Preferably,two or more polynucleotides encoding a delta-4-desaturase are expressed.More preferably, at least one polynucleotide encoding a Coenzyme Adependent delta-4-desaturase and at least one polynucleotide encoding aphospholipid dependent delta-4-desaturase.

Preferably, at least two of the further polynucleotides are expressed.Further, the present invention contemplates the expression of all threefurther polynucleotides. Thus, the method may further compriseexpressing at least one polynucleotide encoding a delta-5-elongase, atleast one polynucleotide encoding a delta-4-desaturase (preferably atleast one polynucleotide for a Coenzyme A dependent delta-4 desaturaseand at least one for a phospholipid dependent delta-4 desaturase), andat least one polynucleotide encoding an omega-3 desaturase.

Moreover, the method of the present invention may further compriseexpressing in the plant at least one polynucleotide encoding adelta-15-desaturase.

In an embodiment, at least one polynucleotide encoding a delta-6elongase from Physcomitrella patens, at least one polynucleotideencoding a delta-12 desaturase from Phythophthora sojae, at least onepolynucleotide encoding a delta-6 desaturase from Ostreococcus tauri, atleast one polynucleotide encoding a delta-6 elongase from Thalassiosirapseudonana, at least one polynucleotide (preferably at least twopolynucleotides) encoding a delta-5 desaturase from Thraustochytrium sp.(preferably from Thraustochytrium sp. ATCC21685), and optionally atleast one polynucleotide (preferably, at least two polynucleotides)encoding a omega-3 desaturase from Pythium irregulare, at least onepolynucleotide encoding a omega-3-desaturase from Phythophthorainfestans, at least one polynucleotide encoding a delta-5 elongase fromOstreococcus tauri, and at least one polynucleotide encoding a delta-4desaturase from Thraustochytrium sp., and at least one polynucleotideencoding a delta-4 desaturase from Pavlova lutheri are expressed.Preferably, at least two polynucleotides encoding a delta-5 desaturasefrom Thraustochytrium sp. (preferably from Thraustochytrium sp.ATCC21685) are expressed. Moreover, it is envisaged to express at leasttwo polynucleotides encoding a omega-3 desaturase from Pythiumirregulare. As set forth elsewhere herein, also variants of theaforementioned polynucleotides can be expressed.

In accordance with the method of the present invention, it is envisagedthat at least one polynucleotide encoding a delta-12-desaturase, atleast one polynucleotide encoding a delta-6-desaturase, at least twopolynucleotides encoding a delta-6-elongase, at least twopolynucleotides encoding a delta-5-desaturase, and optionally at leastthree polynucleotides encoding an omega-3-desaturase, and at least onepolynucleotide encoding a delta-5-elongase, and at least twopolynucleotides encoding a delta-4-desaturase are expressed. Preferably,at least one polynucleotide encoding a Coenzyme A dependentdelta-4-desaturase and at least one polynucleotide encoding aphospholipid dependent delta-4-desaturase are expressed.

In an embodiment, the polynucleotides are expressed in the seeds of theplant.

In accordance with the present invention, the tocopherol content shallbe preferably increased in the seeds of the plant as compared to thetocopherol content in seeds of a control plant, in particular thetocopherol content is increased in the seed oil of the plant as comparedto the seed oil of a control plant.

Preferably, the polynucleotides encoding the elongases and desaturasesreferred to above are recombinant polynucleotides. They may be expressedin a plant by introducing them into the plant by recombinant means suchas Agrobacterium-mediated transformation. Thus, the method may comprisethe steps of introducing and expressing the above-referencedpolynucleotides.

In one embodiment, the method may further comprise the step of selectingfor plants having an increased tocopherol content (as compared to acontrol plant).

In accordance with the present invention, polynucleotides are referredto herein above present on one T-DNA or construct (and thus on the sameT-DNA or construct). Said construct or T-DNA shall be is stablyintegrated in the genome of the plant. In an embodiment, the plant ishomozygous for the T-DNA. In another embodiment, the plant is hemizygousfor the T-DNA. If the plant is homozygous for one T-DNA at one locus,this is nevertheless considered as a single copy herein, i.e. as onecopy. Double copy, as used herein, refers to a plant in which two T-DNAshave been inserted, at one or two loci, and in the hemizygous orhomozygous state.

The present invention also relates to a construct or T-DNA comprisingexpression cassettes for the polynucleotides as set forth in the contextof method of the present invention for increasing the tocopherolcontent.

Preferably, the construct or T-DNA shall comprise expression cassettesfor at least one polynucleotide encoding a delta-12-desaturase, at leastone polynucleotide encoding a delta-6-desaturase, at least onepolynucleotide encoding a delta-6-elongase, and at least onepolynucleotide encoding a delta-5-desaturase, and optionally for atleast one of the further polynucleotides encoding the desaturases orelongases referred to above.

The present invention further concerns the use of the polynucleotides asset forth in the context of the present invention, or of a construct orT-DNA comprising expression cassettes for said polynucleotides forincreasing the tocopherol content of a plant relative to control plants.

The present invention also relates to a plant comprising expressioncassettes for the polynucleotides as referred to in the context of themethod of the present invention for increasing the tocopherol content,or comprising the T-DNA or construct of the present invention.

The present invention also relates to a seed of the plant of the presentinvention. Said seed shall comprise expression cassettes for thepolynucleotides as referred to in the context of the method of thepresent invention for increasing the tocopherol content, or comprisingthe T-DNA or construct of the present invention. In an embodiment, theseed shall comprise an of oil the present invention. The oil isdescribed herein below.

Preferably, the method for increasing the tocopherol content comprisesthe further step of obtaining an oil from the plant, in particular fromthe seeds of the plant. Said oil shall have an increased tocopherolcontent as specified elsewhere herein. In addition, the oil shall havean increased content of VLC-PUFAs. In accordance with the presentinvention, the oil shall be obtained from the plant under conditionswhich maintain the tocopherol content. Such methods are well known inthe art.

The invention also provides methods of producing an oil, wherein the oilhas a high content of tocopherol. In addition, the oil may have a highVLC-PUFA content, in particular a high content of EPA and/or DHA. Inparticularly preferred aspects these methods are for producing acorresponding plant oil. Thus, the invention also provides methods ofproducing an oil.

The invention also provides methods for creating a plant, such that theplant or progeny thereof can be used as a source of an oil having a highcontent of tocopherol. Preferably, the oil further has a high VLC-PUFAcontent, in particular a high content of EPA and/or DHA. Thus, theinvention beneficially also provides methods for the production ofplants having a heritable phenotype of high tocopherol content in seedoil. Further, the plants may have a high VLC-PUFA content in one or moreof their tissues or components, preferably a high content of EPA and/orDHA in seed oil.

DETAILED DESCRIPTION OF THE INVENTION

Various aspects of the invention are hereinafter described in moredetail. The definitions and explantions given in the previous sectionapply accordingly. It is to be understood that the detailed descriptionis not intended to limit the scope of the claims.

Tocopherols are well known in the art. The term “content of tocopherol”preferably refers to the total tocopherol content, i.e. to the sum ofthe amounts of the tocopherols present in the plant, plant part(preferably in the seed) or oil (in particular in seed oil) thereof. Inparticular, the term refers to the sum of the amounts ofalpha-tocopherol, beta-tocopherol, gamma-tocopherol, anddelta-tocopherol. However, it is also envisaged that term refers to theamount of alpha-tocopherol, the amount of beta-tocopherol, and to theamount of gamma-ocopherol, or the amount of delta-tocopherol. In apreferred embodiment, the term refers to the amount of gamma-tocopherol.In another preferred embodiment, the term to the amount ofdelta-tocopherol. Also preferably, the term refers to the amount oftotal tocopherol, gamma-tocopherol and/or delta-tocopherol.

Thus, “tocopherol” in the context of the present invention, preferably,refers to total tocopherol, alpha-tocopherol, beta-tocopherol,gamma-tocopherol, and/or delta-tocopherol. In particular, the termrefers to total tocopherol, gamma-tocopherol, or delta-tocopherol

The term “amount” or “content” preferably refers to the absolute amountor the concentration (preferably, in the plant, more preferably in theseed and most preferably in the seed oil). In an embodiment, the contentof tocopherol is increased in the seed oil of a plant as compared to theseed oil of a control plant.

Increasing the content of tocopherols refers to the increase of thecontent of tocopherols in a plant, or a part, tissue or organ thereof,preferably in the seed, in particular in the oil compared to a controlplant by at least 1%, at least 5%, at least 10%, at least 12% or atleast 15%.

An “increased content” or “high content” of tocopherol as referred toherein preferably refers to a total content of tocopherol in seed oil ofmore than 97 mg/100 g seed oil, in particular of more than 100 mg/100 gseed oil, a content of alpha tocopherol in seed oil of more than 31mg/100 g seed oil, in particular of more than 33 mg/100 g seed oil, acontent of beta tocopherol in seed oil of more than 0.6 mg/100 g seedoil, a content of gamma tocopherol in seed oil of more than 65 mg/100 gseed oil, in particular of more than 70 mg/100 g seed oil, or a contentof delta tocopherol in seed oil of more than 1.4 mg/100 g seed oil, inparticular of more than 1.5 mg/100 g seed oil.

Also, an “increased content” or “high content” of tocopherol as referredto herein preferably refers to a total seed content of tocopherol ofmore than 35 mg/100 g seed, in particular of more than 39 mg/100 g seed,a seed content of alpha tocopherol in seed of more than 12 mg/100 gseed, in particular of more than 13 mg/100 g seed, a seed content ofbeta tocopherol in seed of more than 0.22 mg/100 g seed, a seed contentof gamma tocopherol in seed of more than 25 mg/100 g seed, in particularof more than 26 mg/100 g seed, or a seed content of delta tocopherol inseed of more than 0.45 mg/100 g seed, in particular of more than 0.48mg/100 g seed.

The seeds, in particular the oil, may further comprise a high VLC-PUFA(very long chain polyunsaturated fatty acid) content.

The choice of suitable control plants is a routine part of anexperimental setup and may include corresponding wild type plants orcorresponding plants without the polynucleotides as encoding desaturasesand elongase as referred to herein. The control plant is typically ofthe same plant species or even of the same variety as the plant to beassessed. The control plant may also be a nullizygote of the plant to beassessed. Nullizygotes (or null control plants) are individuals missingthe transgene by segregation. Further, control plants are grown underthe same or essentially the same growing conditions to the growingconditions of the plants of the invention, i.e. in the vicinity of, andsimultaneously with, the plants of the invention. A “control plant” asused herein preferably refers not only to whole plants, but also toplant parts, including seeds and seed parts. The control could also bethe oil from a control plant.

Preferably, the control plant is an isogenic control plant (thus, thecontrol oil e.g. shall be from an isogenic control plant).

The term “polyunsaturated fatty acids (PUFA)” as used herein refers tofatty acids comprising at least two, preferably, three, four, five orsix, double bonds. Moreover, it is to be understood that such fattyacids comprise, preferably from 18 to 24 carbon atoms in the fatty acidchain. More preferably, the term relates to long chain PUFA (VLC-PUFA)having from 20 to 24 carbon atoms in the fatty acid chain. Particularly,polyunsaturated fatty acids in the sense of the present invention areDHGLA 20:3 (8,11,14), ARA 20:4 (5,8,11,14), ETA 20:4 (8,11,14,17), EPA20:5 (5,8,11,14,17), DPA 22:5 (4,7,10,13,16), DPA n-3 (7,10,13,16,19)DHA 22:6 (4,7,10,13,16,19), more preferably, eicosapentaenoic acid (EPA)20:5 (5,8,11,14,17), and docosahexaenoic acid (DHA) 22:6(4,7,10,13,16,19). Thus, it will be understood that most preferably, themethods provided by the present invention pertain to the manufacture ofEPA and/or DHA and/or tocopherol. Moreover, also encompassed are theintermediates of VLC-PUFA which occur during synthesis. Suchintermediates are, preferably, formed from substrates by the desaturase,keto-acyl-CoA-synthase, keto-acyl-CoA-reductase, dehydratase andenoyl-CoA-reductase activity of the polypeptide of the presentinvention. Preferably, substrates encompass LA 18:2 (9,12), GLA 18:3(6,9,12), DHGLA 20:3 (8,11,14), ARA 20:4 (5,8,11,14), eicosadienoic acid20:2 (11,14), eicosatetraenoic acid 20:4 (8,11,14,17), eicosapentaenoicacid 20:5 (5,8,11,14,17). Systematic names of fatty acids includingpolyunsaturated fatty acids, their corresponding trivial names andshorthand notations used according to the present invention are given inthe following table:

Short Short Systematic name Trivial Name hand 1 hand 2 Hexadecanoic acidPalmitic acid 16:0 (Z)-7-Hexadecenoic acid 16:1n-9(Z,Z,Z)-7,10,13-Hexadecatrienoic acid 16:3n-3 Octadecanoic acid Stearicacid 18:0 (Z)-9-Octadecenoic acid Oleic acid 18:1n-9 OA(Z,Z)-9,12-Octadecadienoic acid Linoleic acid 18:2n-6 LA(Z,Z)-6,9-Octadecadienoic acid 18:2n-9 (Z,Z,Z)-9,12,15-Octadecatrienoicacid alpha-Linolenic acid 18:3n-3 ALA (Z,Z,Z)-6,9,12-Octadecatrienoicacid gamma-Linolenic 18:3n-6 GLA acid(Z,Z,Z,Z)-6,9,12,15-Octadecatetraenoic acid Stearidonic acid 18:4n-3 SDAEicosanoic acid Arachidic acid 20:0 (Z)-11-Eicosenoic acid Gondoic acid20:1n-9 (Z,Z)-11,14-Eicosadienoic acid 20:2n-6(Z,Z,Z)-11,14,17-Eicosatrienoic acid 20:3n-3(Z,Z,Z)-8,11,14-Eicosatrienoic acid Dihomo-gamma- 20:3n-6 DHGLAlinolenic acid (Z,Z,Z)-5,8,11-Eicosatrienoic acid Mead acid 20:3n-9(Z,Z,Z,Z)-8,11,14,17-Eicosatetraenoic acid 20:4n-3 ETA(Z,Z,Z,Z)-5,8,11,14-Eicosatetraenoic acid Arachidonic acid 20:4n-6 ARA(Z,Z,Z,Z,Z)-5,8,11,14,17-Eicosapentaenoic acid Timnodonic acid 20:5n-3EPA Docosanoic acid Behenic acid 22:0 (Z)-13-Docosenoic acid Erucic acid22:1n-9 (Z,Z,Z,Z)-7,10,13,16-Docosatetraenoic acid Adrenic acid 22:4n-6DTA (Z,Z,Z,Z,Z)-7,10,13,16,19-Docosapentaenoic acid Clupanodonic acid22:5n-3 DPAn-3 (Z,Z,Z,Z,Z)-4,7,10,13,16-Docosapentaenoic acid Osbondacid 22:5n-6 DPAn-6 (Z,Z,Z,Z,Z,Z)-4,7,10,13,16,19-Docosahexaenoic acid22:6n-3 DHA

The term “cultivating” as used herein refers to maintaining and growingthe transgenic plant under culture conditions which allow the cells toproduce tocopherol in a plant, a seed comprising an increased tocopherolcontent or an oil comprising and increased tocopherol content (ascompared to a control). This implies that the polynucleotides asreferred to herein in connection with the method of the presentinvention are present in the plant. Suitable culture conditions forcultivating the host cell are described in more detail below.

Preferably, the polynucleotides encoding the enzymes as referred toherein are stably integrated into the genome of the plant. Morepreferably, the polynucleotides are present on one T-DNA or constructwhich is stably integrated into the genome of the plant. Thus, they arepreferably present on a single, i.e. the same T-DNA (or construct). Thesame applies to the expression cassettes as referred to herein.Accordingly, the polynucleotides or expression cassttes are preferablycomprised by the same T-DNA.

It is to be understood that more than one copy of the T-DNA (orconstruct) may be present in the plant (e.g. in plants which arehomozygous for the T-DNA (or construct), or in plants in whichAgrobacterium mediated transformation resulted in more than oneintegration event.

The term “obtaining” as used herein encompasses the provision of thecell culture including the host cells and the culture medium or theplant or plant part, particularly the seed, of the current invention, aswell as the provision of purified or partially purified preparationsthereof comprising the tococpherol The plant, plant part or purified orpartially purified preparations may further comprise the polyunsaturatedfatty acid, preferably, ARA, EPA, DHA, in free or in CoA bound form, asmembrane phospholipids or as triacylglyceride esters. More preferably,the PUFA and VLC-PUFA are to be obtained as triglyceride esters, e.g.,in the form of an oil. More details on purification techniques can befound elsewhere herein below.

The term “polynucleotide” according to the present invention refers to adesoxyribonucleic acid or ribonucleic acid. Unless stated otherwise,“polynucleotide” herein refers to a single strand of a DNApolynucleotide or to a double stranded DNA polynucleotide. The length ofa polynucleotide is designated according to the invention by thespecification of a number of basebairs (“bp”) or nucleotides (“nt”).According to the invention, both specifications are usedinterchangeably, regardless whether or not the respective nucleic acidis a single or double stranded nucleic acid. Also, as polynucleotidesare defined by their respective nucleotide sequence, the termsnucleotide/polynucleotide and nucleotide sequence/polynucleotidesequence are used interchangeably, thus that a reference to a nucleicacid sequence also is meant to define a nucleic acid comprising orconsisting of a nucleic acid stretch, the sequence of which is identicalto the nucleic acid sequence.

In particular, the term “polynucleotide” as used in accordance with thepresent invention as far as it relates to a desaturase or elongase generelates to a polynucleotide comprising a nucleic acid sequence whichencodes a polypeptide having desaturase or elongase activity. Preferredpolynucleotides encoding polypeptides having desaturase or elongaseactivity as shown in Table 2 in the Examples section (the SEQ ID NOs ofthe nucleic acid sequences and the polypeptide sequences are given inthe last two columns).

Preferably, the polypeptides encoded by the polynucleotides of thepresent invention having desaturase or elongase activity upon combinedexpression in a plant shall be capable of increasing the content, andthus the amount of tocopherol in a plant in particular, in seeds, seedoils or an entire plant or parts thereof. Whether an increase isstatistically significant can be determined by statistical tests wellknown in the art including, e.g., Student's t-test with aconfidentiality level of at least 90%, preferably of at least 95% andeven more preferably of at least 98%. More preferably, the increase isan increase of the amount of tocopherol of at least 1%, at least 5%, atleast 10%, at least 12% or at least 15% (preferably, by weight) comparedto a control, in particular to the content in seeds, seed oil, crudeoil, or refined oil from a control.

In addition, the polypeptides having desaturase or elongase activityupon combined expression in a plant shall be capable of increasing theamount of PUFA and, in particular, VLC-PUFA in, e.g., seed oils or anentire plant or parts thereof. More preferably, the increase is anincrease of the amount of triglycerides containing VLC-PUFA of at least5%, at least 10%, at least 15%, at least 20% or at least 30% (preferablyby weight) compared to wild-type control, in seeds, seed oil, crude oil,or refined oil from a wildtype control.

Thus, the present invention allows for producing an oil having not onlyan increased tocopherol content as compared to tocopherol content of oilof control plants but also an increased content of PUFA, in particular,of VLC-PUFA.

Preferably, the VLC-PUFA referred to before is a polyunsaturated fattyacid having a C20, C22 or C24 fatty acid body, more preferably EPAand/or DHA. Lipid analysis of oil samples are shown in the accompanyingExamples.

The fatty acid esters with polyunsaturated C20- and/or C22-fatty acidmolecules can be isolated in the form of an oil or lipid, for example,in the form of compounds such as sphingolipids, phosphoglycerides,lipids, glycolipids such as glycosphingolipids, phos-pholipids such asphosphatidylethanolamine, phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol,monoacylglycerides, diacylglycerides, triacylglycerides or other fattyacid esters such as the acetylcoenzyme A esters which comprise thepolyunsaturated fatty acids with at least two, three, four, five or six,preferably five or six, double bonds, from the organisms which were usedfor the preparation of the fatty acid esters. Preferably, they areisolated in the form of their diacylglycerides, triacylglycerides and/orin the form of phosphatidylcholine, especially preferably in the form ofthe triacylglycerides. In addition to these esters, the polyunsaturatedfatty acids are also present in the non-human transgenic organisms orhost cells, preferably in the plants, as free fatty acids or bound inother compounds. The fatty acids are, preferably, produced in boundform. It is possible, with the aid of the polynucleotides andpolypeptides of the present invention, for these unsaturated fatty acidsto be positioned at the sn1, sn2 and/or sn3 position of thetriglycerides which are, preferably, to be produced.

The desaturares and elongases referred to herein are well known in theart.

The term “desaturase” encompasses all enzymatic activities and enzymescatalyzing the desaturation of fatty acids with different lengths andnumbers of unsaturated carbon atom double bonds. Specifically thisincludes delta 4 (d4)-desaturase, preferably catalyzing thedehydrogenation of the 4th and 5th carbon atom; Delta 5 (d5)-desaturasecatalyzing the dehydrogenation of the 5th and 6th carbon atom; Delta 6(d6)-desaturase catalyzing the dehydrogenation of the 6th and 7th carbonatom; Delta 15 (d15)-desaturase catalyzing the dehydrogenation of the15th and 16th carbon atom. An omega 3 (o3) desaturase preferablycatalyzes the dehydrogenation of the n-3 and n-2 carbon atom.

The terms “elongase” encompasses all enzymatic activities and enzymescatalyzing the elongation of fatty acids with different lengths andnumbers of unsaturated carbon atom double bonds. Preferably, the term“elongase” as used herein refers to the activity of an elongase,introducing two carbon molecules into the carbon chain of a fatty acid,preferably in the positions 1, 5, 6, 9, 12 and/or 15 of fatty acids.

In a preferred embodiment, the term “elongase” shall to the activity ofan elongase, introducing two carbon molecules to the carboxyl ends (i.e.position 1) of both saturated and unsaturated fatty acids.

In the studies underlying this invention, enzymes with superiordesaturase and elongase catalytic activities for the increasing thecontent of tocopherol has been provided. Table 2 in the Examples sectionlists preferred polynucleotides encoding for preferred desaturases orelongase to be used in the present invention. Thus, polynucleotidesdesaturases or elongases that can be used in the context of the presentinvention are shown in table 2. As set forth elsewhere herein, alsovariants of the said polynucleotides can be used.

Polynucleotides encoding polypeptides which exhibit delta-6-elongaseactivity have been described in WO2001/059128, WO2004/087902 andWO2005/012316, said documents, describing this enzyme fromPhyscomitrella patens, are incorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-5-desaturaseactivity have been described in WO2002026946 and WO2003/093482, saiddocuments, describing this enzyme from Thraustochytrium sp., areincorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-6-desaturaseactivity have been described in WO2005/012316, WO2005/083093,WO2006/008099 and WO2006/069710, said documents, describing this enzymefrom Ostreococcus tauri, are incorporated herein in their entirety.

In an embodiment, the delta-6-desaturase is a CoA (Coenzyme A)-dependentdelta-6-desaturase.

Polynucleotides encoding polypeptides which exhibit delta-6-elongaseactivity have been described in WO2005/012316, WO2005/007845 andWO2006/069710, said documents, describing this enzyme from Thalassiosirapseudonana, are incorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-12-desaturaseactivity have been described for example in WO2006100241, saiddocuments, describing this enzyme from Phytophthora sojae, areincorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-4-desaturaseactivity have been described for example in WO2004/090123, saiddocuments, describing this enzyme from Euglena gracilis, areincorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-5-elongaseactivity have been described for example in WO2005/012316 andWO2007/096387, said documents, describing this enzyme from Ostreococcustauri, are incorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit omega 3-desaturaseactivity have been described for example in WO2008/022963, saiddocuments, describing this enzyme from Pythium irregulare, areincorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit omega 3-desaturaseactivity have been described for example in WO2005012316 andWO2005083053, said documents, describing this enzyme from Phytophthorainfestans, are incorporated herein in their entirety.

Polynucleotides encoding polypeptides which exhibit delta-4-desaturaseactivity have been described for example in WO2002026946, saiddocuments, describing this enzyme from Thraustochytrium sp., areincorporated herein in their entirety.

Polynucleotides coding for a delta-4 desaturase from Pavlova lutheri aredescribed in WO2003078639 and WO2005007845. These documents areincorporated herein in their entirety, particularly insofar as thedocuments relate to the delta-4 desaturase “PIDES 1” and FIGS. 3a-3d ofWO2003078639 and FIGS. 3a, 3b of WO2005007845, respectively.

Polynucleotides encoding polypeptides which exhibit delta-15-desaturaseactivity have been described for example in WO2010/066703, saiddocuments, describing this enzyme from Cochliobolus heterostrophus C5,are incorporated herein in their entirety.

The polynucleotides encoding the aforementioned polypeptides are hereinalso referred to as “target genes” or “nucleic acid of interest”. Thepolynucleotides are well known in the art. The sequences of saidpolynucleotides can be found in the sequence of the T-DNA disclosed inthe Examples section (see e.g. the sequence of VC-LTM593-1qcz which hasa sequence as shown in SEQ ID NO: 3, see also Table 1). Thepolynucleotide and polypeptide sequences are also given in Table 2 inthe Examples section.

Sequences of preferred polynucleotides for the desaturases and elongasesreferred to herein in connection with the present invention areindicated below. As set forth elsewhere herein, also variants of thepolynucleotides can be used. The polynucleotides encoding fordesaturases and elogases to be used in accordance with the presentinvention can be derived from certain organisms. Preferably, apolynucleotide derived from an organism (e.g from Physcomitrella patens)is codon-optimized. In particular, the polynucleotide shall becodon-optimized for expression in a plant.

The term “codon-optimized” is well understood by the skilled person.Preferably, a codon optimized polynucleotide is a polynucleotide whichis modified by comparison with the nucleic acid sequence in the organismfrom which the sequence originates in that it is adapted to the codonusage in one or more plant species. Typically, the polynucleotide, inparticular the coding region, is adapted for expression in a givenorganism (in particular in a plant) by replacing at least one, or morethan one of codons with one or more codons that are more frequently usedin the genes of that organism (in particular of the plant). Inaccordance with the present invention, a codon optimized variant of aparticular polynucleotide “from an organism” (or “derived from anorganism”) preferably shall be considered to be a polynucleotide derivedfrom said organism.

Preferably, a codon-optimized polynucleotide shall encode for the samepolypeptide having the same sequence as the polypeptide encoded by thenon codon-optimized polynucleotide (i.e. the wild-type sequence). In thestudies underlying the present invention, codon optimizedpolynucleotides were used (for the desaturases). The codon optimizedpolynucleotides are comprised by the T-DNA of the vector having asequence as shown in SEQ ID NO: 3 (see table 1).

The sequences of preferred polynucleotides for the desaturases andelongases and the sequences corresponding polypeptides referred toherein in connection with the present invention are described hereinbelow. Of course variants of polynucleotides and polynucleotides can beused in connection with the present invention (in particular inconnection with the methods, T-DNAs, constructs, plants, seeds, etc.).

Preferably, a delta-6-elongase to be used in accordance with the presentinvention is derived from Physcomitrella patens. A preferred sequence ofsaid delta-6-elongase is shown in SEQ ID NO:258. Preferably, saiddelta-6-elongase is encoded by a polynucleotide derived fromPhyscomitrella patens, in particular, said delta-6-elongase is encodedby a codon-optimized variant thereof. Preferably, the polynucleotideencoding the delta-6-elongase derived from Physcomitrella patens is apolynucleotide having a sequence as shown in nucleotides 1267 to 2139 ofSEQ ID NO: 3. The sequence of this polynucleotide is also shown in SEQID No: 257.

Preferably, a delta-5-desaturase to be used in accordance with thepresent invention is derived from Thraustochytrium sp. Thraustochytriumsp. in the context of the present invention preferably meansThraustochytrium sp. ATCC21685. A preferred sequence of saiddelta-5-desaturase is shown in SEQ ID NO:260. Preferably, saiddelta-5-desaturase is encoded by a polynucleotide derived fromThraustochytrium sp.; in particular, said delta-5-desaturase is encodedby a codon-optimized variant of said polynucleotide. Preferably, thepolynucleotide encoding the delta-5-desaturase derived fromThraustochytrium sp. is a polynucleotide having a sequence as shown innucleotides 3892 to 5211 of SEQ ID NO: 3. The sequence of thispolynucleotide is also shown in SEQ ID No: 259. In accordance with thepresent invention, it is envisaged to express two or morepolynucleotides (i.e. two or more copies of a polynucleotide) encoding adelta-5-desaturase derived from Thraustochytrium sp. (preferably twopolynucleotides). Thus, the T-DNA, construct, plant, seed etc. of thepresent invention shall comprise two (or more) copies of apolynucleotide encoding a delta-5-desaturase derived fromThraustochytrium sp.

Preferably, a delta-6-desaturase to be used in accordance with thepresent invention is derived from Ostreococcus tauri. A preferredsequence of said delta-6-desaturase is shown in SEQ ID NO:262.Preferably, said delta-6-desaturase is encoded by a polynucleotidederived from Ostreococcus tauri; in particular, said delta-6-desaturaseis encoded by a codon-optimized variant of said polynucleotide.Preferably, the polynucleotide encoding the delta-6-desaturase derivedfrom Ostreococcus tauri is a polynucleotide having a sequence as shownin nucleotides 7802 to 9172 of SEQ ID NO: 3. The sequence of thispolynucleotide is also shown in SEQ ID No: 261.

Preferably, a delta-6-elongase to be used in accordance with the presentinvention is derived from Thalassiosira pseudonana. A preferred sequenceof said delta-6-elongase is shown in SEQ ID NO:264. Preferably, saiddelta-6-elongase is encoded by a polynucleotide derived fromThalassiosira pseudonana; in particular, said delta-6-elongase isencoded by a codon-optimized variant of said polynucleotide. Preferably,the polynucleotide encoding the delta-6-elongase derived fromThalassiosira pseudonana is a polynucleotide having a sequence as shownin nucleotides 12099 to 12917 of SEQ ID NO: 3. The sequence of thispolynucleotide is also shown in SEQ ID No: 263. (Thus, thepolynucleotide encoding the delta-6-elongase derived from Thalassiosirapseudonana preferably has a sequence as shown in SEQ ID NO: 263)Preferably, a delta-12-elongase to be used in accordance with thepresent invention is derived from Phytophthora sojae. A preferredsequence of said delta-12-elongase is shown in SEQ ID NO:266.Preferably, said delta-12-elongase is encoded by a polynucleotidederived from Phytophthora sojae; in particular, said delta-12-elongaseis encoded by a codon-optimized variant of said polynucleotide.Preferably, the polynucleotide encoding the delta-12-elongase derivedfrom Phytophthora sojae is a polynucleotide having a sequence as shownin nucleotides 14589 to 15785 of SEQ ID NO: 3. The sequence of thispolynucleotide is also shown in SEQ ID No: 265.

Preferably, a delta-5-elongase to be used in accordance with the presentinvention is derived from Ostreococcus tauri. A preferred sequence ofsaid delta-5-elongase is shown in SEQ ID NO:276. Preferably, saiddelta-5-elongase is encoded by a polynucleotide derived fromOstreococcus tauri; in particular, said delta-5-elongase is encoded by acodon-optimized variant of said polynucleotide. Preferably, thepolynucleotide encoding the delta-5-elongase derived from Ostreococcustauri is a polynucleotide having a sequence as shown in nucleotides38388 to 39290 of SEQ ID NO: 3. The sequence of this polynucleotide isalso shown in SEQ ID No: 275.

Preferably, an omega 3-desaturase to be used in accordance with thepresent invention is derived from Pythium irregulare. A preferredsequence of said omega 3-desaturase is shown in SEQ ID NO:268.Preferably, said omega 3-desaturase is encoded by a polynucleotidederived from Pythium irregulare; in particular, said omega 3-desaturaseis encoded by a codon-optimized variant of said polynucleotide.Preferably, the polynucleotide encoding the omega 3-desaturase derivedfrom Pythium irregulare is a polynucleotide having a sequence as shownin nucleotides 17690 to 18781 of SEQ ID NO: 3. The sequence of thispolynucleotide is also shown in SEQ ID No: 267. In accordance with thepresent invention, it is envisaged to express two or morepolynucleotides (i.e. two or more copies of a polynucleotide) encoding aomega 3-desaturase derived from Pythium irregulare (preferably twopolynucleotides). Thus, the T-DNA, construct, plant, seed etc. of thepresent invention shall comprise two (or more) copies of apolynucleotide encoding a omega 3-desaturase derived from Pythiumirregulare Preferably, an omega 3-desaturase to be used in accordancewith the present invention is derived from Phytophthora infestans. Apreferred sequence of said omega 3-desaturase is shown in SEQ ID NO:270.Preferably, said omega 3-desaturase is encoded by a polynucleotidederived from Phytophthora infestans; in particular, said omega3-desaturase is encoded by a codon-optimized variant of saidpolynucleotide. Preferably, the polynucleotide encoding the omega3-desaturase derived from Phytophthora infestans is a polynucleotidehaving a sequence as shown in nucleotides 20441 to 21526 of SEQ ID NO:3. The sequence of this polynucleotide is also shown in SEQ ID No: 269.

In accordance with the method of the present invention, it is inparticular envisaged to express two or more non-identicalpolynucleotides encoding, preferably non-identical omega 3-desaturasesin the plant. Preferably, at least one polynucleotide encoding an omega3-desaturase from Phytophthora infestans and at least one polynucleotide(in particular two polynucleotides, i.e. two copies of a polynucleotide)encoding an omega 3-desaturase from Pythium irregulare are expressed.

Preferably, a delta-4-desaturase to be used in accordance with thepresent invention is derived from Thraustochytrium sp. A preferredsequence of said delta-4-desaturase is shown in SEQ ID NO:272.Preferably, said delta-4-desaturase is encoded by a polynucleotidederived from Thraustochytrium sp.; in particular, saiddelta-4-desaturase is encoded by a codon-optimized variant of saidpolynucleotide. Preferably, the polynucleotide encoding thedelta-4-desaturase derived from Thraustochytrium sp. is a polynucleotidehaving a sequence as shown in nucleotides 26384 to 27943 of SEQ ID NO:3. The sequence of this polynucleotide is also shown in SEQ ID No: 271.

Preferably, a delta-4-desaturase to be used in accordance with thepresent invention is derived from Pavlova lutheri. A preferred sequenceof said delta-4-desaturase is shown in SEQ ID NO:274. Preferably, saiddelta-4-desaturase is encoded by a polynucleotide derived from Pavlovalutheri; in particular, said delta-4-desaturase is encoded by acodon-optimized variant of said polynucleotide. Preferably, thepolynucleotide encoding the delta-4-desaturase derived from Pavlovalutheri is a polynucleotide having a sequence as shown in nucleotides34360 to 35697 of SEQ ID NO: 3. The sequence of this polynucleotide isalso shown in SEQ ID No: 273.

In accordance with the method of the present invention, it is furtherenvisaged to express two non-identical polynucleotides encoding,preferably non-identical delta-4-desaturases in the plant. Preferably,at least one polynucleotide encoding a delta-4-desaturase fromThraustochytrium sp. and at least one polynucleotide (in particular twopolynucleotides) encoding a delta-4-desaturase from Pavlova lutheri areexpressed.

Preferably, a delta-15-desaturase to be used in accordance with thepresent invention is derived from Cochliobolus heterostrophus.Preferably, said delta-15-desaturase is encoded by a polynucleotidederived from Cochliobolus heterostrophus; in particular, saiddelta-15-desaturase is encoded by a codon-optimized variant of saidpolynucleotide. Preferably, the polynucleotide encoding thedelta-15-desaturase derived from Cochliobolus heterostrophus is apolynucleotide having a sequence as shown in nucleotides 2151 to 3654 ofSEQ ID NO: 9.

As set forth above, the polynucleotide encoding a delta-6-elongase canbe derived from Physcomitrella patens. Moreover, the polynucleotideencoding a delta-6-elongase can be derived from Thalassiosirapseudonana. In particular, it is envisaged in the context of the methodof the present invention to express at least one polynucleotide encodinga delta-6-elongase from Physcomitrella patens and at least onepolynucleotide encoding a delta-6-elongase from Thalassiosira pseudonanain the plant.

A polynucleotide encoding a polypeptide having a desaturase or elongaseactivity as specified above is obtainable or obtained in accordance withthe present invention for example from an organism of genusOstreococcus, Thraustochytrium, Euglena, Thalassiosira, Phytophthora,Pythium, Cochliobolus, Physcomitrella. However, orthologs, paralogs orother homologs may be identified from other species. Preferably, theyare obtained from plants such as algae, for example Isochrysis,Mantoniella, Crypthecodinium, algae/diatoms such as Phaeodactylum,mosses such as Ceratodon, or higher plants such as the Primulaceae suchas Aleuritia, Calendula stellata, Osteospermum spinescens orOsteospermum hyoseroides, microorganisms such as fungi, such asAspergillus, Entomophthora, Mucor or Mortierella, bacteria such asShewanella, yeasts or animals. Preferred animals are nematodes such asCaenorhabditis, insects or vertebrates. Among the vertebrates, thenucleic acid molecules may, preferably, be derived from Euteleostomi,Actinopterygii; Neopterygii; Teleostei; Euteleostei,Protacanthopterygii, Salmoniformes; Salmonidae or Oncorhynchus, morepreferably, from the order of the Salmoniformes, most preferably, thefamily of the Salmonidae, such as the genus Salmo, for example from thegenera and species Oncorhynchus mykiss, Trutta trutta or Salmo truttafario. Moreover, the nucleic acid molecules may be obtained from thediatoms such as the genera Thalassiosira or Phaeodactylum.

Thus, the term “polynucleotide” as used in accordance with the presentinvention further encompasses variants or derivatives of theaforementioned specific polynucleotides representing orthologs, paralogsor other homologs of the polynucleotide of the present invention.Moreover, variants or derivatives of the polynucleotide of the presentinvention also include artificially generated muteins. Said muteinsinclude, e.g., enzymes which are generated by mutagenesis techniques andwhich exhibit improved or altered substrate specificity, or codonoptimized polynucleotides.

Nucleic acid variants or derivatives according to the invention arepolynucleotides which differ from a given reference polynucleotide by atleast one nucleotide substitution, addition and/or deletion. If thereference polynucleotide codes for a protein, the function of thisprotein is conserved in the variant or derivative polynucleotide, suchthat a variant nucleic acid sequence shall still encode a polypeptidehaving a desaturase or elongase activity as specified above. Variants orderivatives also encompass polynucleotides comprising a nucleic acidsequence which is capable of hybridizing to the aforementioned specificnucleic acid sequences, preferably, under stringent hybridizationconditions. These stringent conditions are known to the skilled in theart and can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N. Y. (1989), 6.3.1-6.3.6. A preferred example forstringent hybridization conditions are hybridization conditions in 6×sodium chloride/sodium citrate (=SSC) at approximately 45° C., followedby one or more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C. (inparticular at 65° C.). The skilled worker knows that these hybridizationconditions differ depending on the type of nucleic acid and, for examplewhen organic solvents are present, with regard to the temperature andconcentration of the buffer. For example, under “standard hybridizationconditions” the temperature ranges depending on the type of nucleicacid, between 42° C. and 58° C. in aqueous buffer, with a concentrationof 0.1 to 5×SSC (pH 7.2). If organic solvent is present in theabovementioned buffer, for example 50% formamide, the temperature understandard conditions is approximately 42° C. The hybridization conditionsfor DNA:DNA hybrids are, preferably, 0.1×SSC and 20° C. to 45° C.,preferably between 30° C. and 45° C. The hybridization conditions forDNA:RNA hybrids are, preferably, 0.1×SSC and 30° C. to 55° C.,preferably between 45° C. and 55° C. The abovementioned hybridizationtemperatures are determined for example for a nucleic acid withapproximately 100 bp (=base pairs) in length and a G+C content of 50% inthe absence of formamide. The skilled worker knows how to determine thehybridization conditions required by referring to textbooks such as thetextbook mentioned above, or the following textbooks: Sambrook et al.,“Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames andHiggins (Ed.) 1985, “Nucleic Acids Hybridization: A Practical Approach”,IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991,“Essential Molecular Biology: A Practical Approach”, IRL Press at OxfordUniversity Press, Oxford. In an embodiment, stringent hybridizationconditions encompass hybridization at 65° C. in 1×SSC, or at 42° C. inix SSC and 50% formamide, followed by washing at 65° C. in 0.3×SSC. Inanother embodiment, stringent hybridization conditions encompasshybridization at 65° C. in ix SSC, or at 42° C. in 1×SSC and 50%formamide, followed by washing at 65° C. in 0.1×SSC.

Alternatively, polynucleotide variants are obtainable by PCR-basedtechniques such as mixed oligonucleotide primer based amplification ofDNA, i.e. using degenerated primers against conserved domains of thepolypeptides of the present invention. Conserved domains of thepolypeptide of the present invention may be identified by a sequencecomparison of the nucleic acid sequences of the polynucleotides or theamino acid sequences of the polypeptides of the present invention. As atemplate, DNA or cDNA from bacteria, fungi, plants, or animals may beused. Further, variants include polynucleotides comprising nucleic acidsequences which are at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or at least 99% identical to the nucleicacid coding sequences shown in any one of the T-DNA sequences given inTable 1 of the Examples, and in particular to polynucleotides encodingthe desaturases or elongases referred to above, in particular theelongases and desaturases given in Table 2. E.g., polynucleotides areenvisaged which are at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, at least 98% or at least 99% identical to thepolynucleotide encoding the delta-4-desaturase from Thraustochytrium sp(and thus to a polynucleotide having sequence as shown in nucleotides26384 to 27943 of SEQ ID NO: 3). Of course, a variant as referred toherein must retain the function of the respective enzyme, e.g. a variantof a delta-4-desaturase must retain delta-4-desaturase activity, or avariant of a delta-12-desaturase must retain delta-12-desaturaseactivity.

The percent identity values are, preferably, calculated over the entireamino acid or nucleic acid sequence region. A series of programs basedon a variety of algorithms is available to the skilled worker forcomparing different sequences. In a preferred embodiment, the percentidentity between two amino acid sequences is determined using theNeedleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol.(48):444-453) which has been incorporated into the needle program in theEMBOSS software package (EMBOSS: The European Molecular Biology OpenSoftware Suite, Rice, P., Longden, I., and Bleasby, A, Trends inGenetics 16(6), 276-277, 2000), a BLOSUM62 scoring matrix, and a gapopening penalty of 10 and a gap extension penalty of 0.5. Guides forlocal installation of the EMBOSS package as well as links toWEB-Services can be found at http://emboss.sourceforge.net. A preferred,non-limiting example of parameters to be used for aligning two aminoacid sequences using the needle program are the default parameters,including the EBLOSUM62 scoring matrix, a gap opening penalty of 10 anda gap extension penalty of 0.5. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe needle program in the EMBOSS software package (EMBOSS: The EuropeanMolecular Biology Open Software Suite, Rice, P., Longden, I., andBleasby, A, Trends in Genetics 16(6), 276-277, 2000), using the EDNAFULLscoring matrix and a gap opening penalty of 10 and a gap extensionpenalty of 0.5. A preferred, non-limiting example of parameters to beused in conjunction for aligning two nucleic acid sequences using theneedle program are the default parameters, including the EDNAFULLscoring matrix, a gap opening penalty of 10 and a gap extension penaltyof 0.5. The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the BLAST seriesof programs (version 2.2) of Altschul et al. (Altschul 1990, J. Mol.Biol. 215:403-10). BLAST using desaturase and elongase nucleic acidsequences of the invention as query sequence can be performed with theBLASTn, BLASTx or the tBLASTx program using default parameters to obtaineither nucleotide sequences (BLASTn, tBLASTx) or amino acid sequences(BLASTx) homologous to desaturase and elongase sequences of theinvention. BLAST using desaturase and elongase protein sequences of theinvention as query sequence can be performed with the BLASTp or thetBLASTn program using default parameters to obtain either amino acidsequences (BLASTp) or nucleic acid sequences (tBLASTn) homologous todesaturase and elongase sequences of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST using defaultparameters can be utilized as described in Altschul et al. (Altschul1997, Nucleic Acids Res. 25(17):3389-3402).

Preferred variants of the polynucleotides having a sequence shown in SEQID NO: 257, 259, 261, 263, 265, 267, 269, 271, 273, or 275 are describedherein below.

Preferably, a variant of a polynucleotide encoding a desaturase orelongase as referred to herein is, preferably, a polynucleotidecomprising a nucleic acid sequence selected from the group consistingof:

a) a nucleic acid sequence being at least 70%, 80%, or 90% identical tothe nucleic acid sequence having a nucleotide sequence as shown in SEQID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 273, or 275,b) a nucleic acid sequence encoding a polypeptide which is at least 70%,80, or 90% identical to a polypeptide having an amino acid sequence asshown in SEQ ID NOs: 258, 260, 262, 264, 266, 268, 270, 272, 274, or276, andc) a nucleic acid sequence which is capable of hybridizing understringent conditions to i) a nucleic acid sequence having a nucleotidesequence as shown in SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271,273, or 275, or to ii) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence as shown in SEQ ID NOs: 258, 260, 262,264, 266, 268, 270, 272, 274, or 276.

As set forth above, the polypeptide encoded by said nucleic acid mustretain the function and thus the activity of the respective enzyme. Forexample, the polypeptide having a sequence as shown in SEQ ID NO: 270has omega-3-desaturase activity. Accordingly, the variant thispolypeptide also shall have omega-3-desaturase activity.

Thus, a polynucleotide encoding a desaturase or elongase as referred toherein is, preferably, a polynucleotide comprising a nucleic acidsequence selected from the group consisting of:

a) a nucleic acid sequence having a nucleotide sequence as shown in SEQID NO: 257, 259, 261, 263, 265, 267, 269, 271, 273, or 275,b) a nucleic acid sequence encoding a polypeptide having an amino acidsequence as shown in SEQ ID NO: 258, 260, 262, 264, 266, 268, 270, 272,274, or 276c) a nucleic acid sequence being at least 70%, 80%, or 90% identical tothe nucleic acid sequence having a nucleotide sequence as shown in SEQID NOs: 257, 259, 261, 263, 265, 267, 269, 271, 273, or 275,d) a nucleic acid sequence encoding a polypeptide which is at least 70%,80, or 90% identical to a polypeptide having an amino acid sequence asshown in SEQ ID NOs: 258, 260, 262, 264, 266, 268, 270, 272, 274, or276, ande) a nucleic acid sequence which is capable of hybridizing understringent conditions to i) a nucleic acid sequence having a nucleotidesequence as shown in SEQ ID NOs: 257, 259, 261, 263, 265, 267, 269, 271,273, or 275, or to ii) a nucleic acid sequence encoding a polypeptidehaving an amino acid sequence as shown in SEQ ID NOs: 258, 260, 262,264, 266, 268, 270, 272, 274, or 276.

The event LBFLFK comprises two T-DNA insertions, the insertions beingdesignated LBFLFK Locus 1 and LBFLFK Locus 2. Plants comprising thisinsertion were generated by transformation with the T-DNA vector havinga sequence as shown in SEQ ID NO: 3.

Sequencing of the insertions present in the plant revealed that eachlocus contained a point mutation in a coding sequence resulting in asingle amino acid exchange. The mutations did not affect the function ofthe genes. Locus 1 has a point mutation in the coding sequence for thedelta-12 desaturase from Phythophthora sojae (d12Des(Ps)). The resultingpolynucleotide has a sequence as shown in SEQ ID NO: 324. Saidpolynucleotide encodes a polypeptide having a sequence as shown in SEQID NO: 325. Locus 2 has a point mutation in the coding sequence for thedelta-4 desaturase from Pavlova lutheri (d4Des(PI)). The resultingpolynucleotide has a sequence as shown in SEQ ID NO: 326. Saidpolynucleotide encodes a polypeptide having a sequence as shown in SEQID NO: 327. The aforementioned polynucleotides are considered asvariants of the polynucleotide encoding the delta-12 desaturase fromPhythophthora sojae and the polynucleotide encoding the delta-4desaturase from Pavlova lutheri. The polynucleotides are considered asvariants and can be used in the context of the present invention.

A polynucleotide comprising a fragment of any nucleic acid, particularlyof any of the aforementioned nucleic acid sequences, is also encompassedas a polynucleotide of the present invention. The fragments shall encodepolypeptides which still have desaturase or elongase activity asspecified above. Accordingly, the polypeptide may comprise or consist ofthe domains of the polypeptide of the present invention conferring thesaid biological activity. A fragment as meant herein, preferably,comprises at least 50, at least 100, at least 250 or at least 500consecutive nucleotides of any one of the aforementioned nucleic acidsequences or encodes an amino acid sequence comprising at least 20, atleast 30, at least 50, at least 80, at least 100 or at least 150consecutive amino acids of any one of the aforementioned amino acidsequences.

The variant polynucleotides or fragments referred to above, preferably,encode polypeptides retaining desaturase or elongase activity to asignificant extent, preferably, at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80% or at least 90% of the desaturase or elongase activity exhibited byany of the polypeptides encoded by T-DNA given in the accompanyingExamples (in particular of the desaturases or elongases listed in Table1 and 2).

In order to express the polynucleotides encoding the desaturases orelongases as set forth in connection with the present invention, thepolynucleotides shall be operably linked to expression controlsequences. Preferably, the expression control sequences are heterologouswith respect to the polynucleotides operably linked thereto. It is to beunderstood that each polynucleotide is operably linked to an expressioncontrol sequence.

The term “expression control sequence” as used herein refers to anucleic acid sequence which is capable of governing, i.e. initiating andcontrolling, transcription of a nucleic acid sequence of interest, inthe present case the nucleic sequences recited above. Such a sequenceusually comprises or consists of a promoter or a combination of apromoter and enhancer sequences. Expression of a polynucleotidecomprises transcription of the nucleic acid molecule, preferably, into atranslatable mRNA. Additional regulatory elements may includetranscriptional as well as translational enhancers. The followingpromoters and expression control sequences may be, preferably, used inan expression vector according to the present invention. The cos, tac,trp, tet, trp-tet, Ipp, lac, Ipp-lac, laclq, T7, T5, T3, gal, trc, ara,SP6, A-PR or A-PL promoters are, preferably, used in Gram-negativebacteria. For Gram-positive bacteria, promoters amy and SPO2 may beused. From yeast or fungal promoters ADC1, AOX1r, GAL1, MFα, AC, P-60,CYC1, GAPDH, TEF, rp28, ADH are, preferably, used. For animal cell ororganism expression, the promoters CMV-, SV40-, RSV-promoter (Roussarcoma virus), CMV-enhancer, SV40-enhancer are preferably used. Fromplants the promoters CaMV/35S (Franck 1980, Cell 21: 285-294], PRP1(Ward 1993, Plant. Mol. Biol. 22), SSU, OCS, lib4, usp, STLS1, B33, nosor the ubiquitin or phaseolin promoter. Also preferred in this contextare inducible promoters, such as the promoters described in EP 0388186A1 (i.e. a benzylsulfonamide-inducible promoter), Gatz 1992, Plant J.2:397-404 (i.e. a tetracyclin-inducible promoter), EP 0335528 A1 (i.e. aabscisic-acid-inducible promoter) or WO 93/21334 (i.e. a ethanol- orcyclohexenol-inducible promoter).

Further suitable plant promoters are the promoter of cytosolic FBPase orthe ST-LSI promoter from potato (Stockhaus 1989, EMBO J. 8, 2445), thephosphoribosyl-pyrophosphate amidotransferase promoter from Glycine max(Genbank accession No. U87999) or the node-specific promoter describedin EP 0249676 A1. Particularly preferred are promoters which enable theexpression in tissues which are involved in the biosynthesis of fattyacids. Also particularly preferred are seed-specific promoters such asthe USP promoter in accordance with the practice, but also otherpromoters such as the LeB4, DC3, phaseolin or napin promoters. Furtherespecially preferred promoters are seed-specific promoters which can beused for monocotyledonous or dicotyledonous plants and which aredescribed in U.S. Pat. No. 5,608,152 (napin promoter from oilseed rape),WO 98/45461 (oleosin promoter from Arabidopsis, U.S. Pat. No. 5,504,200(phaseolin promoter from Phaseolus vulgaris), WO 91/13980 (Bce4 promoterfrom Brassica), by Baeumlein et al., Plant J., 2, 2, 1992:233-239 (LeB4promoter from a legume), these promoters being suitable for dicots. Thefollowing promoters are suitable for monocots: Ipt-2 or Ipt-1 promoterfrom barley (WO 95/15389 and WO 95/23230), hordein promoter from barleyand other promoters which are suitable and which are described in WO99/16890. In principle, it is possible to use all natural promoterstogether with their regulatory sequences, such as those mentioned above,for the novel process. Likewise, it is possible and advantageous to usesynthetic promoters, either additionally or alone, especially when theymediate a seed-specific expression, such as, for example, as describedin WO 99/16890. Preferably, the polynucleotides encoding the desaturasesand elongases as referred to herein are expressed in the seeds of theplants. In a particular embodiment, seed-specific promoters are utilizedin accordance with the present invention. In a particular preferredembodiment the polynucleotides encoding the desaturares or elongases areoperably linked to expression control sequences used for the expressionof the desaturases and elongases in the Examples section (see e.g. thepromoters used for expressing the elongases and desaturases inVC-LTM593-1qcz rc. The sequence of this vector is shown in SEQ ID NO: 3,see also Table 1 in the Examples section).

The term “operatively linked” as used herein means that the expressioncontrol sequence and the nucleic acid of interest are linked so that theexpression of the said nucleic acid of interest can be governed by thesaid expression control sequence, i.e. the expression control sequenceshall be functionally linked to the said nucleic acid sequence to beexpressed. Accordingly, the expression control sequence and, the nucleicacid sequence to be expressed may be physically linked to each other,e.g., by inserting the expression control sequence at the 5′end of thenucleic acid sequence to be expressed. Alternatively, the expressioncontrol sequence and the nucleic acid to be expressed may be merely inphysical proximity so that the expression control sequence is capable ofgoverning the expression of at least one nucleic acid sequence ofinterest. The expression control sequence and the nucleic acid to beexpressed are, preferably, separated by not more than 500 bp, 300 bp,100 bp, 80 bp, 60 bp, 40 bp, 20 bp, 10 bp or 5 bp. Preferredpolynucleotides of the present invention comprise, in addition to apromoter, a terminator sequence operatively linked to the nucleic acidsequence of interest. Thereby, an expression cassette is formed.

The term “terminator” as used herein refers to a nucleic acid sequencewhich is capable of terminating transcription. These sequences willcause dissociation of the transcription machinery from the nucleic acidsequence to be transcribed. Preferably, the terminator shall be activein plants and, in particular, in plant seeds. Suitable terminators areknown in the art and, preferably, include polyadenylation signals suchas the SV40-poly-A site or the tk-poly-A site or one of the plantspecific signals indicated in Loke et al. (Loke 2005, Plant Physiol 138,pp. 1457-1468), downstream of the nucleic acid sequence to be expressed.

In a preferred embodiment, the polynucleotides encoding the desaturasesor elongase referred to herein are recombinant.

The invention furthermore relates to recombinant nucleic acid moleculescomprising at least one nucleic acid sequence which codes for apolypeptide having desaturase and/or elongase activity which is modifiedby comparison with the nucleic acid sequence in the organism from whichthe sequence originates in that it is adapted to the codon usage in oneor more plant species.

For the purposes of the invention “recombinant” means with regard to,for example, a nucleic acid sequence, an expression cassette (=geneconstruct) or a vector comprising the nucleic acid sequences used in theprocess according to the invention or a host cell transformed with thenucleic acid sequences, expression cassette or vector used in theprocess according to the invention, all those constructions broughtabout by recombinant methods in which either the nucleic acid sequence,or a genetic control sequence which is operably linked with the nucleicacid sequence, for example a promoter, or are not located in theirnatural genetic environment or have been modified by recombinantmethods.

The definitions given herein above preferably apply to the following:

As set forth above, the present invention relates to a method forincreasing the tocopherol content of a plant relative to a controlplant, comprising expressing in a plant at least one polynucleotideencoding a delta-12-desaturase, at least one polynucleotide encoding adelta-6-desaturase, at least one polynucleotide encoding adelta-6-elongase, and at least one polynucleotide encoding adelta-5-desaturase. In an embodiment, the method further comprises theexpression of at least one polynucleotide encoding anomega-3-desaturase, at least one polynucleotide encoding adelta-5-elongase, and/or at least one polynucleotide encoding adelta-4-desaturase (for more details regarding the method of the presentinvention, see section “SUMMARY OF THE INVENTION”, the definitions andexplanations apply accordingly). Preferably, the polynucleotides areexpressed from expression cassettes.

The invention is also concerned with providing polynucleotides as setforth in connection with the method of the present invention, constructsor T-DNAs for establishing high tocopherol content in plants or partsthereof, particularly in plant oils.

The construct or T-DNA shall comprise expression cassettes for thepolynucleotides as set forth in the context of the method of the presentinvention for increasing the tocopherol content. The construct or T-DNAcan be used in connection with the method the present invention. In anembodiment, said construct or T-DNA is introduced into the plant forexpressing the said polynucleotides (for increasing the tocopherolcontent).

Accordingly, the present invention relates to a construct or T-DNAcomprising at least one expression cassette for a delta-12-desaturase,at least one expression cassette for a delta-6-desaturase, at least oneexpression cassette for a delta-6-elongase, and at least one expressioncassette for a delta-5-desaturase.

An expression cassette for expression of a gene (herein also referred toas target gene) shall comprise the polynucleotide encoding therespective enzyme (i.e. a desaturase or an elongase) operatively linkedto a promoter (expression control sequence). Preferably, the expressioncassette further comprises a terminator. Preferably, the terminator isdownstream of the polynucleotide encoding the desaturase or elongase.

In an embodiment, the construct or T-DNA further comprises at least oneexpression cassette for an omega-3-desaturase.

In an embodiment, the construct or T-DNA further comprises at least oneexpression cassette for a delta-5-elongase.

In an embodiment, the construct or T-DNA further comprises at least oneexpression cassette for a delta-4-desaturase.

In an embodiment, the construct or T-DNA further comprises at least oneexpression cassette for a delta-15-desaturase.

In a preferred embodiment, the construct or T-DNA further comprises atleast one expression cassette for an omega-3-desaturase, at least oneexpression cassette for a delta-5-elongase, and at least one expressioncassette for a delta-4-desaturase (preferably at least one for aCoenzyme A dependent delta-4 desaturase and at least one for aphospholipid dependent delta-4 desaturase).

In a particularly preferred embodiment, the T-DNA or construct comprisesat least one expression cassette for a delta-12-desaturase, at least oneexpression cassette for a delta-6-desaturase, at least two expressioncassettes for a delta-6-elongase, at least two expression cassettes fora delta-5-desaturase, and optionally at least three expression cassettesfor an omega-3-desaturase, and at least one expression cassette for adelta-5-elongase, and at least two expression cassettes for adelta-4-desaturase (preferably for one CoA (Coenzyme A)-dependent D4Desand for one Phospholipid-dependent d4Des.)

In another preferred embodiment, the T-DNA or construct comprises atleast one expression cassette for a delta-6 elongase from Physcomitrellapatens, at least one expression cassette for a delta-12 desaturase fromPhythophthora sojae, at least one expression cassette for a delta-6desaturase from Ostreococcus tauri, at least one expression cassette fora delta-6 elongase from Thalassiosira pseudonana, at least oneexpression cassette (in particular at least two) expression cassette(s)for a delta-5 desaturase from Thraustochytrium sp., and optionally atleast one expression cassette (in particular at least two) expressioncassette(s) for an omega-3 desaturase from Pythium irregulare, at leastone expression cassette for an omega-3-desaturase from Phythophthorainfestans, at least one expression cassette for a delta-5 elongase fromOstreococcus tauri, and at least one expression cassette for a delta-4desaturase from Thraustochytrium sp., and at least one expressioncassette for a delta-4 desaturase from Pavlova lutheri.

Also preferably, the T-DNA or construct comprises the sequence of theT-DNA in the T-DNA vector VC-LTM593-1qcz described in the Examplessection. This vector comprises a sequence shown in SEQ ID NO: 3.

Thus, the invention provides a T-DNA for expression of a target gene ina plant, wherein the T-DNA comprises a left and a right border elementand at least one expression cassette comprising a promoter, operativelylinked thereto a target gene, and downstream thereof a terminator (andthus at least the expression cassette referred to above), wherein thelength of the T-DNA, measured from left to right border element andcomprising the target gene, has a length of at least 30000 bp. In anembodiment, the expression cassette is separated from the closest borderof the T-DNA by a separator of at least 500 bp length.

In an embodiment, the T-DNA or construct of the present invention maycomprise a separator between the expression cassettes encoding for thedesaturases or elongases referred to above. Preferably, the expressioncassettes are separated from each other by a separator of at least 100base pairs, preferably of 100 to 200 base pairs. Thus, there is aseparator between each expression cassette.

The invention thus provides nucleic acids, i.e. polynucleotides. Apolynucleotide according to the present invention is or comprises aT-DNA or construct according to the present invention. Thus, a T-DNAaccording to the present invention is a polynucleotide, preferably aDNA, and most preferably a double stranded DNA. A “T-DNA” according tothe invention is a nucleic acid capable of eventual integration into thegenetic material (genome) of a plant. The skilled person understandsthat for such integration a transformation of respective plant materialis required, preferred transformation methods and plant generationmethods are described herein.

According to the invention also provided are nucleic acids comprising aT-DNA or construct as defined according to the present invention. Forexample, a T-DNA of the present invention may be comprised in a circularnucleic acid, e.g. a plasmid, such that an additional nucleic acidsection is present between the left and right border elements, i.e.“opposite” of the expression cassette(s) according to the presentinvention. Such circular nucleic acid may be mapped into a linear formusing an arbitrary starting point, e.g. such that the definition “leftborder element-expression cassette-right border element-additionalnucleic acid section opposite of the expression cassette” defines thesame circular nucleic acid as the definition “expression cassette-rightborder element-additional nucleic acid section opposite of theexpression cassette-left border element”. The additional nucleic acidsection preferably comprises one or more genetic elements forreplication of the total nucleic acid, i.e. the nucleic acid moleculecomprising the T-DNA and the additional nucleic acid section, in one ormore host microorganisms, preferably in a microorganism of genusEscherichia, preferably E. coli, and/or Agrobacterium. Preferable hostmicroorganisms are described below in more detail. Such circular nucleicacids comprising a T-DNA of the present invention are particularlyuseful as transformation vectors; such vectors and are described belowin more detail.

The polynucleotides as referred to herein are preferably expressed in aplant after introducing them into a plant. Thus, the method of thepresent invention may also comprise the step of introducing thepolynucleotides into the plant. Preferably, the polynucleotides areintroduced into the plant by transformation, in particular byAgrobacterium-mediated transformation. In an embodiment, the plants aretransformed with a construct or T-DNA comprising the polynucleotidesand/or expression cassette as set forth in connect with the presentinvention. Thus, it is envisaged that the plant is (has been)transformed with a T-DNA or construct of the present invention. Theconstruct or T-DNA used for the introduction, preferably comprises allpolynucleotides to be expressed. Thus, a single construct or T-DNA shallbe used for transformation.

The T-DNA or construct length is, thus, preferably large, i.e. may havea minimum length of at least 15000 bp, preferably more than 30000 bp,more preferably at least 40000 bp, even more preferably at least 50000bp and most preferably at least 60000 bp. Preferably, the length of theT-DNA is in a range of any of the aforementioned minimum lengths to120000 bp, more preferably in a range of any of the aforementionedminimum lengths to 100000 bp, even more preferably in a range of any ofthe aforementioned minimum lengths to 90000 bp, even more preferably ina range of any of the aforementioned minimum lengths to 80000 bp. Withsuch minimum lengths it is possible to introduce a number of genes inthe form of expression cassettes such that each individual gene isoperably liked to at least one promoter and at least one terminator.

In an embodiment, in 3′ direction of the T-DNA left border element or in5′ direction of the T-DNA right border element, a separator is presentsetting the respective border element apart from the expression cassettecomprising the target gene. The separator in 3′ direction of the T-DNAleft border element does not necessarily have the same length and/orsequence as the separator in 5′ direction of the T-DNA right borderelement, as long as both separators suffice to the further requirementsgiven below.

In another embodiment, the expression cassettes are separated from eachother by a separator of at least 100 base pairs, preferably of 100 to200 base pairs. Thus, there is a separator between the expressioncassettes.

The separator or spacer is a section of DNA predominantly defined by itslength. Its function is to separate a target gene from the T-DNA's leftor right border, respectively. Introducing a separator effectivelyseparates the gene of interest from major influences exerted by theneighbouring genomic locations after insertion of the T-DNA into agenomic DNA. For example it is commonly believed that not all genomicloci are equally suitable for expression of a target gene, and that thesame gene under the control of the same promoter and terminator may beexpressed in different intensity in plants depending on the region ofintegration of the target gene (and its corresponding promoter andterminator) in the plant genome. It is generally believed that differentregions of a plant genome are accessible with differing ease fortranscription factors and/or polymerase enzymes, for example due tothese regions being tightly wound around histones and/or attached to thechromosomal backbone (cf. for example Deal et al., Curr Opin Plant Biol.April 2011; 14(2): 116-122) or other scaffold material (cf. e.g. FukudaY., Plant Mol Biol. 1999 March; 39(5): 1051-62). The mechanism ofachieving the abovementioned benefits by the T-DNA of the presentinvention is not easily understood, so it is convenient to think of thespacer as a means for physically providing a buffer to compensate forstrain exerted by DNA winding by neighbouring histones or chromosomalbackbone or other scaffold attached regions. As a model it can bethought that to transcribe a target gene, the DNA has to be partiallyunwound. If neighbouring regions of the target gene resist suchunwinding, for example because they are tightly wound around histones orotherwise attached to a scaffold or backbone such that rotation ofnucleic acid strands is limited, the spacer allows to distribute thestrain created by the unwinding attempt over a longer stretch of nucleicacid, thereby reducing the force required for unwinding at the targetgene.

In an embodiment, the separator has a length of at least 500 bp. Theseparator, thus, can be longer than 500 bp, and preferably is at least800 bp in length, more preferably at least 1000 bp. Longer spacers allowfor even more physical separation between the target gene and thenearest genomic flanking region.

In another embodiment, the spacer has a length of at least 100 bp.Preferably, the spacer has a length of 100 to 200 base pairs.

The separator preferably has a sequence devoid of matrix or scaffoldattachment signals. Preferably, the separator or spacer does notcomprise more than once for a length of 500 bp, preferably not more thanonce for a length of 1000 bp, a 5-tuple which occurs in the spacers for20 or more times, summarized over all spacers given in the examples.Those 5-tuples are, in increasing frequency in the spacers given in theexamples: AGCCT, CGTAA, CTAAC, CTAGG, GTGAC, TAGGC, TAGGT, AAAAA, AACGC,TTAGC, ACGCT, GCTGA, ACGTT, AGGCT, CGTAG, CTACG, GACGT, GCTTA, AGCTT,CGCTA, TGACG, ACGTG, AGCTG, CACGT, CGTGA, CGTTA, AGCGT, TCACG, CAGCT,CGTCA, CTAGC, GCGTC, TTACG, GTAGC, TAGCG, TCAGC, TAGCT, AGCTA, GCTAG,ACGTA, TACGT. By reducing the frequency of occurrence of one or more ofthe aforelisted 5-tuples compared to the separators or spacers, afurther increase in expression of a target gene in the T-DNA can beachieved.

The separator may contain a selectable marker. A selectable marker is anucleic acid section whose presence preferably can be verified in seedwithout having to wait for the sprouting or full growth of the plant.Preferably the selectable marker conveys a phenotypical property to seedor to a growing plant, for example herbicide tolerance, coloration, seedsurface properties (e.g. wrinkling), luminescence or fluorescenceproteins, for example green fluorescent protein or luciferase. If forexhibiting the phenotypical feature an expression of a marker gene isrequired, then the separator correspondingly comprises the marker geneas a selectable marker, preferably in the form of an expressioncassette. Inclusion of a selectable marker in the separator isparticularly advantageous since the marker allows easy discard ofnon-transformant plant material. Also, in such unexpected case where theT-DNA integrates in a location of the plant genome where the lengthand/or nucleobase composition of the spacer is insufficient to overcomegene silencing effects caused by the neighbouring genomic DNA, theselectable marker allows easy discard of such unfortunately badlyperforming exceptional transformants. Thus, preferably the separatorcomprises an expression cassette for expression of an herbicidetolerance gene. Such separator greatly reduces the chance of having tocultivate a transformant where silencing effects are so strong that eventhe expression of the selectable marker gene is greatly reduced or fullyinhibited. According to the invention, the separator preferably does notcomprise a desaturase or elongase gene, and also preferably does notcomprise a promoter or operatively linked to a desaturase or elongasegene. Thus, the T-DNA of the present invention in preferred embodimentsis useful for effective separation of the desaturase and elongase genesessential for the production of VLC-PUFAs from any influence of effectscaused by neighbouring genomic plant DNA.

For increasing the tocopherol content (and for the production ofVLC-PUFAs) in plants, the invention also provides a construct or a T-DNAcomprising the coding sequences (in particular of the desaturases andelogases) as given in Table 1 and 2 in the examples, preferablycomprising the coding sequences (in particular of the desaturases andelogases) and promoters as given in Table 1 in the examples, morepreferably the coding sequences (in particular of the desaturases andelongases) and promoters and terminators as given in Table 1 in theexamples, and most preferably the expression cassettes for thedesaturases and elongases as referred to in the context of the method ofpresent invention as present in VC-LTM593-1qcz rc (see Examples section,SEQ ID NO: 3).

The present invention furthermore relates to a plant comprising thepolynucleotides as referred to herein in the context of the method ofthe present invention for increasing the tocopherol content, or theT-DNA or construct of the present invention. Furthermore, the presentinvention relates to a seed of the plant. Said seed shall comprised thesaid polynucleotides. In an embodiment, the said polynucleotides arecomprised by the same T-DNA.

In addition, the present invention relates to Brassica plant, or a seedthereof, having in increased tocopherol content a compared to a controlplant, in particular having an increased tocopherol content the seeds ascompared to the seeds of control plants. In an embodiment, said plant isa Brassica napus plant. Said plant shall be transgenic.

In a preferred embodiment, the seed of the present invention shallcomprise an oil as described herein below in more detail.

The plant of the invention shall comprise one or more T-DNA or constructof the present invention. Thus, the plant shall comprise at least T-DNAor construct of the present invention. Moreover, it is envisaged thatthe plant of the present invention comprises the polynucleotidesencoding desaturases as set forth in the context of the method of thepresent invention of increasing the tocopherol content.

Preferably, the T-DNA or construct comprised by the plant comprises oneor more expression cassettes encoding for one or more d6Des (delta 6desaturase), one or more d6Elo (delta 6 elongase), one or more d5Des(delta 5 desaturase), or one more d12Des (delta 12 desaturase). In anembodiment, the T-DNA or construct comprised by the plant of the presentinvention, further comprises expression cassettes for one or more o3Des(omega 3 desaturase), one or more d5Elo (delta 5 elongase) and/or one ormore d4Des (delta 4 desaturase), preferably for at least one CoA(Coenzyme A)-dependent D4Des and one Phospholipid-dependent d4Des.

Three desaturase genes are particularly prone to gene dosage effects(also called “copy number effects”), such that increasing the number ofexpression cassettes comprising these respective genes leads to astronger increase in VLC-PUFA levels in plant oils than increasing thenumber of expression cassettes of other genes. These genes are the genescoding for delta-12-desaturase activity, for delta-6-desaturase activityand omega-3-desaturase activity. It is to be understood that where theT-DNA of the present invention comprises more than one expressioncassette comprising a gene of the same function, these genes do not needto be identical concerning their nucleic acid sequence or thepolypeptide sequence encoded thereby, but should be functional homologs.Thus, for example, to make use of the gene dosage effect describedherein a T-DNA according to the present invention may comprise, inaddition to optionally a multiplicity of genes coding fordelta-6-desaturases and/or omega-3-desaturases, two, three, four or moreexpression cassettes each comprising a gene coding for adelta-12-desaturase, wherein the delta-12-desaturase polypeptides codedby the respective genes differ in their amino acid sequence. Likewise, aT-DNA of the present invention may comprise, in addition to optionally amultiplicity of genes coding for delta-12-desaturases and/oromega-3-desaturases, two, three, four or more expression cassettes eachcomprising a gene coding for a delta-6-desaturase, wherein thedelta-6-desaturase polypeptides coded by the respective genes differ intheir amino acid sequence, or a T-DNA of the present invention maycomprise, in addition to optionally a multiplicity of genes coding fordelta-12-desaturases and/or delta-6-desaturases, two, three, four ormore expression cassettes each comprising a gene coding for aomega-3-desaturase, wherein the omega-3-desaturase polypeptides coded bythe respective genes differ in their amino acid sequence.

According to the invention, the T-DNA, construct or plant may alsocomprise, instead of one or more of the aforementioned coding sequences,a functional homolog thereof. A functional homolog of a coding sequenceis a sequence coding for a polypeptide having the same metabolicfunction as the replaced coding sequence. For example, a functionalhomolog of a delta-5-desaturase would be another delta-5-desaturase, anda functional homolog of a delta-5-elongase would be anotherdelta-5-elongase. The functional homolog of a coding sequence preferablycodes for a polypeptide having at least 40% sequence identity to thepolypeptide coded for by the corresponding coding sequence given Table 1of the examples, more preferably at least 41%, more preferably at least46%, more preferably at least 48%, more preferably at least 56%, morepreferably at least 58%, more preferably at least 59%, more preferablyat least 62%, more preferably at least 66%, more preferably at least69%, more preferably at least 73%, more preferably at least 75%, morepreferably at least 77%, more preferably at least 81%, more preferablyat least 84%, more preferably at least 87%, more preferably at least90%, more preferably at least 92%, more preferably at least 95%, morepreferably at least 96%, more preferably at least 97%, more preferablyat least 98% and even more preferably at least 99%. Likewise, afunctional homolog of a promoter is a sequence for startingtranscription of a coding sequence located within 500 bp for a proximalpromoter or, for a distal promoter, within 3000 bp distant from thepromoter TATA box closest to the coding sequence. Again, a functionalhomolog of a plant seed specific promoter is another plant seed specificpromoter. The functional homolog of a terminator, correspondingly, is asequence for ending transcription of a nucleic acid sequence.

The Examples describe a particularly preferred T-DNA sequence. Theskilled person understands that the coding sequences, promoters andterminators described therein can be replaced by their functionalhomologs. However, the Examples also describe that according to theinvention, certain combinations of promoters and coding sequences, orcertain combinations of promoters driving the expression of theircorresponding coding sequences, or certain coding sequences orcombinations thereof are particularly advantageous; such combinations orindividual coding sequences should according to the invention not bereplaced by functional homologs of the respective element (here: codingsequence or promoter). Preferred promoter-coding sequence-terminatorcombinations are shown in Table 1.

A T-DNA or construct of the present invention may comprise two or moregenes, preferably all genes, susceptible to a gene dosage effect. Asdescribed herein, it is advantageous for achieving high conversionefficiencies of certain enzymatic activities, e.g. delta-12-desaturase,delta-6-desaturase and/or omega-3-desaturase activity, to introduce morethan one gene coding for an enzyme having the desired activity into aplant cell. When introducing T-DNA into plant cells, generallytransformation methods involving exposition of plant cells tomicroorganisms are employed, e.g. as described herein. As eachmicroorganism may comprise more than one nucleic acid comprising a T-DNAof the present invention, recombinant plant cells are frequentlyobtained comprising two or more T-DNAs of the present inventionindependently integrated into the cell's genetic material. Thus, bycombining genes susceptible to a gene dosage effect on one construct fortransformation allows to easily exploit the independence oftransformations to achieve a higher frequency of multiple insertions ofsuch T-DNAs. This is particularly useful for transformation methodsrelying on co-transformation to keep the size of each construct to betransformed low.

The invention accordingly also provides a construct comprising a T-DNAaccording to the present invention, wherein the construct preferably isa vector for transformation of a plant cell by microorganism-mediatedtransformation, preferably by Agrobacterium-mediated transformation.Correspondingly, the invention also provides a transformingmicroorganism comprising one T-DNA according to the present invention,preferably as a construct comprising said T-DNA. Preferably themicroorganism is of genus Agrobacterium, preferably a disarmed strainthereof, and preferably of species Agrobacterium tumefaciens or, evenmore preferably, of species Agrobacterium rhizogenes. Correspondingstrains are for example described in WO06024509A2, and methods for planttransformation using such microorganisms are for example described inWO13014585A1. These WO publications are incorporated herein in theirentirety, because they contain valuable information about the creation,selection and use of such microorganisms.

The term “vector”, preferably, encompasses phage, plasmid, viral vectorsas well as artificial chromosomes, such as bacterial or yeast artificialchromosomes. Moreover, the term also relates to targeting constructswhich allow for random or site-directed integration of the targetingconstruct into genomic DNA. Such target constructs, preferably, compriseDNA of sufficient length for either homolgous or heterologousrecombination as described in detail below. The vector encompassing thepolynucleotide of the present invention, preferably, further comprisesselectable markers for propagation and/or selection in a host. Thevector may be incorporated into a host cell by various techniques wellknown in the art. If introduced into a host cell, the vector may residein the cytoplasm or may be incorporated into the genome. In the lattercase, it is to be understood that the vector may further comprisenucleic acid sequences which allow for homologous recombination orheterologous insertion. Vectors can be introduced into prokaryotic oreukaryotic cells via conventional transformation or transfectiontechniques. The terms “transformation” and “transfection”, conjugationand transduction, as used in the present context, are intended tocomprise a multiplicity of prior-art processes for introducing foreignnucleic acid (for example DNA) into a host cell, including calciumphosphate, rubidium chloride or calcium chloride co-precipitation,DEAE-dextran-mediated transfection, lipofection, natural competence,carbon-based clusters, chemically mediated transfer, electroporation orparticle bombardment. Suitable methods for the transformation ortransfection of host cells, including plant cells, can be found inSambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., ColdSpring Harbor Laboratory, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989) and other laboratory manuals, such as Methodsin Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.:Gartland and Davey, Humana Press, Totowa, N.J. Alternatively, a plasmidvector may be introduced by heat shock or electroporation techniques.Should the vector be a virus, it may be packaged in vitro using anappropriate packaging cell line prior to application to host cells.

Preferably, the vector referred to herein is suitable as a cloningvector, i.e. replicable in microbial systems. Such vectors ensureefficient cloning in bacteria and, preferably, yeasts or fungi and makepossible the stable transformation of plants. Those which must bementioned are, in particular, various binary and co-integrated vectorsystems which are suitable for the T DNA-mediated transformation. Suchvector systems are, as a rule, characterized in that they contain atleast the vir genes, which are required for the Agrobacterium-mediatedtransformation, and the sequences which delimit the T-DNA (T-DNAborder). These vector systems, preferably, also comprise furthercis-regulatory regions such as promoters and terminators and/orselection markers with which suitable transformed host cells ororganisms can be identified. While co-integrated vector systems have virgenes and T-DNA sequences arranged on the same vector, binary systemsare based on at least two vectors, one of which bears vir genes, but noT-DNA, while a second one bears T-DNA, but no vir gene. As aconsequence, the last-mentioned vectors are relatively small, easy tomanipulate and can be replicated both in E. coli and in Agrobacterium.These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks,pGreen series. Preferably used in accordance with the invention are Binl9, pBI101, pBinAR, pGPTV and pCAM BIA. An overview of binary vectors andtheir use can be found in Hellens et al, Trends in Plant Science (2000)5, 446-451. Furthermore, by using appropriate cloning vectors, thepolynucleotides can be introduced into host cells or organisms such asplants or animals and, thus, be used in the transformation of plants,such as those which are published, and cited, in: Plant MolecularBiology and Biotechnology (CRC Press, Boca Raton, Fla.), chapter 6/7,pp. 71-119 (1993); F. F. White, Vectors for Gene Transfer in HigherPlants; in: Transgenic Plants, vol. 1, Engineering and Utilization, Ed.:Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et al., Techniquesfor Gene Transfer, in: Transgenic Plants, vol. 1, Engineering andUtilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143;Potrykus 1991, Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205-225.

More preferably, the vector of the present invention is an expressionvector. In such an expression vector, i.e. a vector which comprises thepolynucleotide of the invention having the nucleic acid sequenceoperatively linked to an expression control sequence (also called“expression cassette”) allowing expression in plant cells or isolatedfractions thereof.

Most important, the invention also provides a plant or seed thereof,comprising, integrated in its genome, a construct or T-DNA of thepresent invention.

Thus, the construct or T-DNA shall be stably integrated into the genomeof the plant or plant cell. The present invention, thus, relates to aplant comprising the T-DNA or construct of the present invention.

Such T-DNA or construct preferably allows for the expression of allgenes required for increasing the tocopherol content in plants andparticularly in the seeds thereof, particularly in oilseed plants, andmost beneficially in plants or seeds of family Brassicaceae, preferablyof genus Brassica and most preferably of a species comprising a genomeof one or two members of the species Brassica oleracea, Brassica nigraand Brassica rapa, thus preferably of the species Brassica napus,Brassica carinata, Brassica juncea, Brassica oleracea, Brassica nigra orBrassica rapa. Particularly preferred according to the invention areplants and seeds of the species Brassica napus and Brassica carinata.

The plants of the present invention are necessarily transgenic, i.e.they comprise genetic material not present in corresponding wild typeplant or arranged differently in corresponding wild type plant, forexample differing in the number of genetic elements. For example, theplants of the present invention comprise promoters also found in wildtype plants, but the plants of the present invention comprise suchpromoter operatively linked to a coding sequence such that thiscombination of promoter and coding sequence is not found in thecorresponding wild type plant. Accordingly, the polynucleotide encodingfor the desaturases or elongases shall be recombinant polynucleotides.

The plants and seeds of the present invention differ from hithertoproduced plants in their production of a high content of tocopherol (andpreferably of VLC-PUFAs), see Examples. In particular, the combinationsof polynucleotides encoding the elongases or desaturases as set forth inconnection with the method of the present invention, the constructs andT-DNAs of the present invention allow for the generation of transformantplants (also called “recombinant plants”) and seeds thereof with a hightransformation frequency, with a high stability of T-DNA insertions overmultiple generations of self-fertilized plants, unchanged or unimpairedphenotypical and agronomic characteristics, with high amounts andconcentration of tocopherol, and with high amounts and concentration ofVLC-PUFAs, particularly EPA and/or DHA, in the oil of populations ofsuch transformed plants and their corresponding progeny.

Unless stated otherwise, a plant of the present invention comprising aT-DNA or construct of the present invention can also be a plantcomprising a part of a T-DNA or construct of the present invention,where such part is sufficient for the production of a desaturase and/orelongase coded for in the corresponding full T-DNA or construct of thepresent invention. Such plants most preferably comprise at least onefull T-DNA of the present invention in addition to the part of a T-DNAof the present invention as defined in the previous sentence. Suchplants are hereinafter also termed “partial double copy” plants. EventLBFDAU is an example of a plant comprising a part of a T-DNA of thepresent invention, and still being a plant of the present invention. Inone embodiment the T DNA is a full T-DNA.

Preferred plants of the present invention comprise one or more T-DNA(s)or construct(s) of the present invention comprising expression cassettescomprising, one or more genes encoding for one or more d5Des, one ormore d6Elo, one or more d6Des, and one or more d12Des. In oneembodiment, at least one T-DNA or vector further comprises (an)expression cassette(s) which comprises one or more genes encoding forone or more d5Elo, one or more o3Des, one or more d15Des, and/or one ormore D4Des, preferably for at least one CoA-dependent D4Des and onePhospholipid-dependent d4Des. In one embodiment, the T-DNA or T-DNAscomprise one or more expression cassettes encoding d6Elo(Tp_GA) and/ord6Elo(Pp_GA). d6Elo(Tp_GA) is a Delta-6 elongase from Thalassiosirapseudonana, d6Elo(Pp_GA) is a Delta-6 elongase from Physcomitrellapatens.

Preferably, the plant (or plant cell) of the present invention is anoilseed crop plant (or an oilseed crop plant cell). More preferably,said oilseed crop is selected from the group consisting of flax (Linumsp.), rapeseed (Brassica sp.), soybean (Glycine and Soja sp.), sunflower(Helianthus sp.), cotton (Gossypium sp.), corn (Zea mays), olive (Oleasp.), safflower (Carthamus sp.), cocoa (Theobroma cacoa), peanut(Arachis sp.), hemp, camelina, crambe, oil palm, coconuts, groundnuts,sesame seed, castor bean, lesquerella, tallow tree, sheanuts, tungnuts,kapok fruit, poppy seed, jojoba seeds and perilla. Preferred plants tobe used for introducing the polynucleotide or T-DNA of the invention areplants which are capable of synthesizing fatty acids, such as alldicotyledonous or monocotyledonous plants, algae or mosses. Preferredplants are selected from the group of the plant familiesAdelotheciaceae, Anacardiaceae, Arecaceae, Asteraceae, Apiaceae,Betulaceae, Boraginaceae, Brassicaceae, Bromeliaceae, Caricaceae,Cannabaceae, Convolvulaceae, Chenopodiaceae, Compositae,Crypthecodiniaceae, Cruciferae, Cucurbitaceae, Ditrichaceae,Elaeagnaceae, Ericaceae, Euphorbiaceae, Fabaceae, Geraniaceae,Gramineae, Juglandaceae, Lauraceae, Leguminosae, Linaceae, Malvaceae,Moringaceae, Marchantiaceae, Onagraceae, Olacaceae, Oleaceae,Papaveraceae, Piperaceae, Pedaliaceae, Poaceae, Solanaceae,Prasinophyceae or vegetable plants or ornamentals such as Tagetes.Examples which may be mentioned are the following plants selected fromthe group consisting of: Adelotheciaceae such as the generaPhyscomitrella, such as the genus and species Physcomitrella patens,Anacardiaceae such as the genera Pistacia, Mangifera, Anacardium, forexample the genus and species Pistacia vera [pistachio], Mangifer indica[mango] or Anacardium occidentale [cashew], Asteraceae, such as thegenera Calendula, Carthamus, Centaurea, Cichorium, Cynara, Helianthus,Lactuca, Locusta, Tagetes, Valeriana, for example the genus and speciesCalendula officinalis [common marigold], Carthamus tinctorius[safflower], Centaurea cyanus [cornflower], Cichorium intybus [chicory],Cynara scolymus [artichoke], Helianthus annus [sunflower], Lactucasativa, Lactuca crispa, Lactuca esculenta, Lactuca scariola L. ssp.sativa, Lactuca scariola L. var. integrata, Lactuca scariola L. var.integrifolia, Lactuca sativa subsp. romana, Locusta communis, Valerianalocusta [salad vegetables], Tagetes lucida, Tagetes erecta or Tagetestenuifolia [african or french marigold], Apiaceae, such as the genusDaucus, for example the genus and species Daucus carota [carrot],Betulaceae, such as the genus Corylus, for example the genera andspecies Corylus avellana or Corylus colurna [hazelnut], Boraginaceae,such as the genus Borago, for example the genus and species Boragoofficinalis [borage], Brassicaceae, such as the genera Brassica,Melanosinapis, Sinapis, Arabadopsis, for example the genera and speciesBrassica napus, Brassica rapa ssp. [oilseed rape], Sinapis arvensisBrassica juncea, Brassica juncea var. juncea, Brassica juncea var.crispifolia, Brassica juncea var. foliosa, Brassica nigra, Brassicasinapioides, Melanosinapis communis [mustard], Brassica oleracea [fodderbeet] or Arabidopsis thaliana, Bromeliaceae, such as the genera Anana,Bromelia (pineapple), for example the genera and species Anana comosus,Ananas ananas or Bromelia comosa [pineapple], Caricaceae, such as thegenus Carica, such as the genus and species Carica papaya [pawpaw],Cannabaceae, such as the genus Cannabis, such as the genus and speciesCannabis sativa [hemp], Convolvulaceae, such as the genera Ipomea,Convolvulus, for example the genera and species Ipomoea batatus, Ipomoeapandurata, Convolvulus batatas, Convolvulus tiliaceus, Ipomoeafastigiata, Ipomoea tiliacea, Ipomoea triloba or Convolvulus panduratus[sweet potato, batate], Chenopodiaceae, such as the genus Beta, such asthe genera and species Beta vulgaris, Beta vulgaris var. altissima, Betavulgaris var. Vulgaris, Beta maritima, Beta vulgaris var. perennis, Betavulgaris var. conditiva or Beta vulgaris var. esculenta [sugarbeet],Crypthecodiniaceae, such as the genus Crypthecodinium, for example thegenus and species Cryptecodinium cohnii, Cucurbitaceae, such as thegenus Cucurbita, for example the genera and species Cucurbita maxima,Cucurbita mixta, Cucurbita pepo or Cucurbita moschata [pumpkin/squash],Cymbellaceae such as the genera Amphora, Cymbella, Okedenia,Phaeodactylum, Reimeria, for example the genus and species Phaeodactylumtricornutum, Ditrichaceae such as the genera Ditrichaceae, Astomiopsis,Ceratodon, Chrysoblastella, Ditrichum, Distichium, Eccremidium,Lophidion, Philibertiella, Pleuridium, Saelania, Trichodon,Skottsbergia, for example the genera and species Ceratodon antarcticus,Ceratodon columbiae, Ceratodon heterophyllus, Ceratodon purpureus,Ceratodon purpureus, Ceratodon purpureus ssp. convolutus, Ceratodon,purpureus spp. stenocarpus, Ceratodon purpureus var. rotundifolius,Ceratodon ratodon, Ceratodon stenocarpus, Chrysoblastella chilensis,Ditrichum ambiguum, Ditrichum brevisetum, Ditrichum crispatissimum,Ditrichum difficile, Ditrichum falcifolium, Ditrichum flexicaule,Ditrichum giganteum, Ditrichum heteromallum, Ditrichum lineare,Ditrichum lineare, Ditrichum montanum, Ditrichum montanum, Ditrichumpallidum, Ditrichum punctulatum, Ditrichum pusillum, Ditrichum pusillumvar. tortile, Ditrichum rhynchostegium, Ditrichum schimperi, Ditrichumtortile, Distichium capillaceum, Distichium hagenii, Distichiuminclinatum, Distichium macounii, Eccremidium floridanum, Eccremidiumwhiteleggei, Lophidion strictus, Pleuridium acuminatum, Pleuridiumalternifolium, Pleuridium holdridgei, Pleuridium mexicanum, Pleuridiumravenelii, Pleuridium subulatum, Saelania glaucescens, Trichodonborealis, Trichodon cylindricus or Trichodon cylindricus var. oblongus,Elaeagnaceae such as the genus Elaeagnus, for example the genus andspecies Olea europaea [olive], Ericaceae such as the genus Kalmia, forexample the genera and species Kalmia latifolia, Kalmia angustifolia,Kalmia microphylla, Kalmia polifolia, Kalmia occidentalis, Cistuschamaerhodendros or Kalmia lucida [mountain laurel], Euphorbiaceae suchas the genera Manihot, Janipha, Jatropha, Ricinus, for example thegenera and species Manihot utilissima, Janipha manihot, Jatrophamanihot, Manihot aipil, Manihot dulcis, Manihot manihot, Manihotmelanobasis, Manihot esculenta [manihot] or Ricinus communis [castor-oilplant], Fabaceae such as the genera Pisum, Albizia, Cathormion,Feuillea, Inga, Pithecolobium, Acacia, Mimosa, Medicajo, Glycine,Dolichos, Phaseolus, Soja, for example the genera and species Pisumsativum, Pisum arvense, Pisum humile [pea], Albizia berteriana, Albiziajulibrissin, Albizia lebbeck, Acacia berteriana, Acacia littoralis,Albizia berteriana, Albizzia berteriana, Cathormion berteriana, Feuilleaberteriana, Inga fragrans, Pithecellobium berterianum, Pithecellobiumfragrans, Pithecolobium berterianum, Pseudalbizzia berteriana, Acaciajulibrissin, Acacia nemu, Albizia nemu, Feuilleea julibrissin, Mimosajulibrissin, Mimosa speciosa, Sericanrda julibrissin, Acacia lebbeck,Acacia macrophylla, Albizia lebbek, Feuilleea lebbeck, Mimosa lebbeck,Mimosa speciosa [silk tree], Medicago sativa, Medicago falcata, Medicagovaria [alfalfa], Glycine max Dolichos soja, Glycine gracilis, Glycinehispida, Phaseolus max, Soja hispida or Soja max [soybean], Funariaceaesuch as the genera Aphanorrhegma, Entosthodon, Funaria, Physcomitrella,Physcomitrium, for example the genera and species Aphanorrhegmaserratum, Entosthodon attenuatus, Entosthodon bolanderi, Entosthodonbonplandii, Entosthodon californicus, Entosthodon drummondii,Entosthodon jamesonii, Entosthodon leibergii, Entosthodon neoscoticus,Entosthodon rubrisetus, Entosthodon spathulifolius, Entosthodon tucsoni,Funaria americana, Funaria bolanderi, Funaria calcarea, Funariacalifornica, Funaria calvescens, Funaria convoluta, Funaria flavicans,Funaria groutiana, Funaria hygrometrica, Funaria hygrometrica var.arctica, Funaria hygrometrica var. calvescens, Funaria hygrometrica var.convoluta, Funaria hygrometrica var. muralis, Funaria hygrometrica var.utahensis, Funaria microstoma, Funaria microstoma var. obtusifolia,Funaria muhlenbergii, Funaria orcuttii, Funaria plano-convexa, Funariapolaris, Funaria ravenelii, Funaria rubriseta, Funaria serrata, Funariasonorae, Funaria sublimbatus, Funaria tucsoni, Physcomitrellacalifornica, Physcomitrella patens, Physcomitrella readeri,Physco-mitrium australe, Physcomitrium californicum, Physcomitriumcollenchymatum, Physcomitrium coloradense, Physcomitrium cupuliferum,Physcomitrium drummondii, Physcomitrium eurystomum, Physcomitriumflexifolium, Physcomitrium hookeri, Physcomitrium hookeri var. serratum,Physcomitrium immersum, Physcomitrium kellermanii, Physcomitriummegalocarpum, Physcomitrium pyriforme, Physcomitrium pyriforme var.serratum, Physcomitrium rufipes, Physcomitrium sandbergii, Physcomitriumsubsphaericum, Physcomitrium washingtoniense, Geraniaceae, such as thegenera Pelargonium, Cocos, Oleum, for example the genera and speciesCocos nucifera, Pelargonium grossularioides or Oleum cocois [coconut],Gramineae, such as the genus Saccharum, for example the genus andspecies Saccharum officinarum, Juglandaceae, such as the genera Juglans,Wallia, for example the genera and species Juglans regia, Juglansailanthifolia, Juglans sieboldiana, Juglans cinerea, Wallia cinerea,Juglans bixbyi, Juglans californica, Juglans hindsii, Juglansintermedia, Juglans jamaicensis, Juglans major, Juglans microcarpa,Juglans nigra or Wallia nigra [walnut], Lauraceae, such as the generaPersea, Laurus, for example the genera and species Laurus nobilis [bay],Persea americana, Persea gratissima or Persea persea [avocado],Leguminosae, such as the genus Arachis, for example the genus andspecies Arachis hypogaea [peanut], Linaceae, such as the genera Linum,Adenolinum, for example the genera and species Linum usitatissimum,Linum humile, Linum austriacum, Linum bienne, Linum angustifolium, Linumcatharticum, Linum flavum, Linum grandiflorum, Adenolinum grandiflorum,Linum lewisii, Linum narbonense, Linum perenne, Linum perenne var.lewisii, Linum pratense or Linum trigynum [linseed], Lythrarieae, suchas the genus Punica, for example the genus and species Punica granatum[pomegranate], Malvaceae, such as the genus Gossypium, for example thegenera and species Gossypium hirsutum, Gossypium arboreum, Gossypiumbarbadense, Gossypium herbaceum or Gossypium thurberi [cotton],Marchantiaceae, such as the genus Marchantia, for example the genera andspecies Marchantia berteroana, Marchantia foliacea, Marchantiamacropora, Musaceae, such as the genus Musa, for example the genera andspecies Musa nana, Musa acuminata, Musa paradisiaca, Musa spp. [banana],Onagraceae, such as the genera Camissonia, Oenothera, for example thegenera and species Oenothera biennis or Camissonia brevipes [eveningprimrose], Palmae, such as the genus Elacis, for example the genus andspecies Elaeis guineensis [oil palm], Papaveraceae, such as the genusPapaver, for example the genera and species Papaver orientale, Papaverrhoeas, Papaver dubium [poppy], Pedaliaceae, such as the genus Sesamum,for example the genus and species Sesamum indicum [sesame], Piperaceae,such as the genera Piper, Artanthe, Peperomia, Steffensia, for examplethe genera and species Piper aduncum, Piper amalago, Piperangustifolium, Piper auritum, Piper betel, Piper cubeba, Piper longum,Piper nigrum, Piper retrofractum, Artanthe adunca, Artanthe elongata,Peperomia elongata, Piper elongatum, Steffensia elongata [cayennepepper], Poaceae, such as the genera Hordeum, Secale, Avena, Sorghum,Andropogon, Holcus, Panicum, Oryza, Zea (maize), Triticum, for examplethe genera and species Hordeum vulgare, Hordeum jubatum, Hordeummurinum, Hordeum secalinum, Hordeum distichon, Hordeum aegiceras,Hordeum hexastichon, Hordeum hexastichum, Hordeum irregulare, Hordeumsativum, Hordeum secalinum [barley], Secale cereale [rye], Avena sativa,Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida[oats], Sorghum bicolor, Sorghum halepense, Sorghum saccharatum, Sorghumvulgare, Andropogon drummondii, Holcus bicolor, Holcus sorghum, Sorghumaethiopicum, Sorghum arundinaceum, Sorghum caffrorum, Sorghum cernuum,Sorghum dochna, Sorghum drummondii, Sorghum durra, Sorghum guineense,Sorghum lanceolatum, Sorghum nervosum, Sorghum saccharatum, Sorghumsubglabrescens, Sorghum verticilliflorum, Sorghum vulgare, Holcushalepensis, Sorghum miliaceum, Panicum militaceum [millet], Oryzasativa, Oryza latifolia [rice], Zea mays [maize], Triticum aestivum,Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha,Triticum sativum or Triticum vulgare [wheat], Porphyridiaceae, such asthe genera Chroothece, Flintiella, Petrovanella, Porphyridium, Rhodella,Rhodosorus, Vanhoeffenia, for example the genus and species Porphyridiumcruentum, Proteaceae, such as the genus Macadamia, for example the genusand species Macadamia intergrifolia [macadamia], Prasinophyceae such asthe genera Nephroselmis, Prasinococcus, Scherffelia, Tetraselmis,Mantoniella, Ostreococcus, for example the genera and speciesNephroselmis olivacea, Prasinococcus capsulatus, Scherffelia dubia,Tetraselmis chui, Tetraselmis suecica, Mantoniella squamata,Ostreococcus tauri, Rubiaceae such as the genus Cofea, for example thegenera and species Cofea spp., Coffea arabica, Coffea canephora orCoffea liberica [coffee], Scrophulariaceae such as the genus Verbascum,for example the genera and species Verbascum blattaria, Verbascumchaixii, Verbascum densiflorum, Verbascum lagurus, Verbascumlongifolium, Verbascum lychnitis, Verbascum nigrum, Verbascum olympicum,Verbascum phlomoides, Verbascum phoenicum, Verbascum pulverulentum orVerbascum thapsus [mullein], Solanaceae such as the genera Capsicum,Nicotiana, Solanum, Lycopersicon, for example the genera and speciesCapsicum annuum, Capsicum annuum var. glabriusculum, Capsicum frutescens[pepper], Capsicum annuum [paprika], Nicotiana tabacum, Nicotiana alata,Nicotiana attenuata, Nicotiana glauca, Nicotiana langsdorffii, Nicotianaobtusifolia, Nicotiana quadrivalvis, Nicotiana repanda, Nicotianarustica, Nicotiana sylvestris [tobacco], Solanum tuberosum [potato],Solanum melongena [eggplant], Lycopersicon esculentum, Lycopersiconlycopersicum, Lycopersicon pyriforme, Solanum integrifolium or Solanumlycopersicum [tomato], Sterculiaceae, such as the genus Theobroma, forexample the genus and species Theobroma cacao [cacao] or Theaceae, suchas the genus Camellia, for example the genus and species Camelliasinensis [tea]. In particular preferred plants to be used as transgenicplants in accordance with the present invention are oil fruit cropswhich comprise large amounts of lipid compounds, such as peanut, oilseedrape, canola, sunflower, safflower, poppy, mustard, hemp, castor-oilplant, olive, sesame, Calendula, Punica, evening primrose, mullein,thistle, wild roses, hazelnut, almond, macadamia, avocado, bay,pumpkin/squash, linseed, soybean, pistachios, borage, trees (oil palm,coconut, walnut) or crops such as maize, wheat, rye, oats, triticale,rice, barley, cotton, cassava, pepper, Tagetes, Solanaceae plants suchas potato, tobacco, eggplant and tomato, Vicia species, pea, alfalfa orbushy plants (coffee, cacao, tea), Salix species, and perennial grassesand fodder crops.

Preferred plants according to the invention are oil crop plants such aspeanut, oilseed rape, canola, sunflower, safflower, poppy, mustard,hemp, castor-oil plant, olive, Calendula, Punica, evening primrose,pumpkin/squash, linseed, soybean, borage, trees (oil palm, coconut).Especially preferred are sunflower, safflower, tobacco, mullein, sesame,cotton, pumpkin/squash, poppy, evening primrose, walnut, linseed, hemp,thistle or safflower. Very especially preferred plants are plants suchas safflower, sunflower, poppy, evening primrose, walnut, linseed, orhemp, or most preferred, plants of family Brassicaceae.

Most preferably, the plant of the present invention is a plant found inthe “Triangle of U”, i.e. a plant of genus Brassica: Brassica napus (AACC genome; n=19) is an amphidiploid plant of the Brassica genus but isthought to have resulted from hybridization of Brassica rapa (AA genome;n=10) and Brassica oleracea (CC genome; n=9). Brassica juncea (AA BBgenome; n=18) is an amphidiploid plant of the Brassica genus that isgenerally thought to have resulted from the hybridization of Brassicarapa and Brassica nigra (BB genome; n=8). Under some growing conditions,B. juncea may have certain superior traits to B. napus. These superiortraits may include higher yield, better drought and heat tolerance andbetter disease resistance. Brassica carinata (BB CC genome; n=17) is anamphidiploid plant of the Brassica genus but is thought to have resultedfrom hybridization of Brassica nigra and Brassica oleracea. Under somegrowing conditions, B. carinata may have superior traits to B. napus.Particularly, B. carinata allows for an increase in VLC-PUFAconcentrations by at least 20% compared to B. napus when transformedwith the same T-DNA.

The plant of the present invention preferably is a “Canola” plant.Canola is a genetic variation of rapeseed developed by Canadian plantbreeders specifically for its oil and meal attributes, particularly itslow level of saturated fat. Canola herein generally refers to plants ofBrassica species that have less than 2% erucic acid (Delta 13-22:1) byweight in seed oil and less than 30 micromoles of glucosinolates pergram of oil-free meal. Typically, canola oil may include saturated fattyacids known as palmitic acid and stearic acid, a monounsaturated fattyacid known as oleic acid, and polyunsaturated fatty acids known aslinoleic acid and linolenic acid. Canola oil may contain less than about7% (w/w) total saturated fatty acids (mostly palmitic acid and stearicacid) and greater than 40% (w/w) oleic acid (as percentages of totalfatty acids). Traditionally, canola crops include varieties of Brassicanapus and Brassica rapa. Preferred plants of the present invention arespring canola (Brassica napus subsp. oleifera var. annua) and wintercanola (Brassica napus subsp. oleifera var. biennis). Furthermore acanola quality Brassica juncea variety, which has oil and meal qualitiessimilar to other canola types, has been added to the canola crop family(U.S. Pat. No. 6,303,849, to Potts et al., issued on Oct. 16, 2001; U.S.Pat. No. 7,423,198, to Yao et al.; Potts and Males, 1999; all of whichare incorporated herein by reference). Likewise it is possible toestablish canola quality B. carinata varieties by crossing canolaquality variants of Brassica napus with Brassica nigra and appropriatelyselecting progeny thereof, optionally after further back-crossing withB. carinata, B. napus and/or B. nigra.

The invention also provides a plant or seed thereof of familyBrassicaceae, preferably of genus Brassica, with a genotype that confersa heritable phenotype of seed oil VLC-PUFA content, obtainable orobtained from progeny lines prepared by a method comprising the steps of

i) crossing a plant of family Brassicaceae, preferably of genusBrassica, most preferably of genus Brassica napus, Brassica oleracea,Brassica nigra or Brassica carinata, said plant comprising a combinationof polynucleotides encoding for desaturases or elongases as set forth inthe context of the method of the present invention, a construct or T-DNAof the present invention and/or part of such construct or T-DNA, with aparent plant of family Brassicaceae, preferably of genus Brassica, mostpreferably of genus Brassica napus, Brassica oleracea, Brassica nigra orBrassica carinata, said plant not comprising said T-DNA and/or partthereof, to yield a F1 hybrid,ii) selfing the F1 hybrid for at least one generation, andiii) identifying the progeny of step (ii) comprising the combination ofpolynucleotides, the construct, T-DNA of the present invention capableof producing seed comprising an increased tocopherol content as comparedto a control plant. In an embodiment, an increased tocopherol content isa tocopherol content as disclosed elsewhere herein.

In an embodiment, the produced seed comprise VLC-PUFA such that thecontent of all VLC-PUFA downstream of 18:1 n-9 is at least 40% (w/w) ofthe total seed fatty acid content at an oil content of 40% (w/w), orpreferably the content of EPA is at least 8%, or at least 12% (w/w)and/or the content of DHA is at least 1% (w/w) of the total seed fattyacid content at an oil content of 40% (w/w).

In an embodiment, the produced seed comprise VLC-PUFA such that thecontent of EPA is at least 8%, or at least 12%. (w/w).

In an embodiment, the content of DHA is at least 1% (w/w) of the totalseed fatty acid content.

This method allows for effectively incorporation of genetic material ofother members of family Brassicaceae, preferably of genus Brassica, intothe genome of a plant comprising the polynucleotides as set forth in thecontext of the method of the present invention, a T-DNA, or construct ofthe present invention. The method is particularly useful for combiningthe polynucleotides, the T-DNA and/or the construct with geneticmaterial responsible for beneficial traits exhibited in other members offamily Brassicaceae. Beneficial traits of other members of familyBrassicaceae are exemplarily described herein, other beneficial traitsor genes and/or regulatory elements involved in the manifestation of abeneficial trait may be described elsewhere.

The parent plant not comprising the said polynucleotides, the T-DNA orthe construct of the present invention or part thereof preferably is anagronomically elite parent. In particular, the present invention teachesthe transfer of heterologous material from a plant or seed of thepresent invention to a different genomic background, for example adifferent variety or species.

In particular, the invention teaches the transfer of the T-DNA or partthereof (the latter is particularly relevant for those plants of thepresent invention which comprise, in addition to a full T-DNA orconstruct of the present invention, also a part of a T-DNA or constructof the present invention, said part preferably comprising at least oneexpression cassette, the expression cassette preferably comprising agene coding for a desaturase or elongase, preferably adelta-12-desaturase, delta-6-desaturase and/or omega-3-desaturase) intoa species of genus Brassica carinata, or to introduce genetic materialfrom Brassica carinata or Brassica nigra into the plants of the presentinvention comprising the T-DNA of the present invention and/or a part ortwo or more parts thereof. According to the invention, genes of Brassicanigra replacing their homolog found in Brassica napus or added inaddition to the homolog found in Brassica napus are particularly helpfulin further increasing the amount of VLC-PUFAs in plant seeds and oilsthereof.

Also, the invention teaches novel plant varieties comprising thepolynucleotides encoding for the desaturases or elongases as set forthin the context of the method of the present invention, the construct orT-DNA and/or part thereof of the present invention. Such varieties can,by selecting appropriate mating partners, be particularly adapted e.g.to selected climatic growth conditions, herbicide tolerance, stressresistance, fungal resistance, herbivore resistance, increased orreduced oil content or other beneficial features. It is particularlybeneficial to provide plants of the present invention wherein the oilcontent thereof at harvest is lower than that of corresponding wild typeplants of the same variety, such as to increase the total tocopherolcontent (and to improve VLC-PUFA amounts) in the oil of said plants ofthe present invention and/or tocopherol concentration (and VLC-PUFAconcentrations) in said oil.

Also, the invention provides a method for creating a plant with agenotype that confers a heritable phenotype of tocopherol content (inparticular an increased content in the seed oil), obtainable or obtainedfrom progeny lines prepared by a method comprising the steps of

i) crossing a transgenic plant of the invention with a parent plant notcomprising the polynucleotides encoding for the desaturases or elongasesas set forth in the context of the method of the present invention, theconstruct or T-DNA of the present invention or part thereof, said parentplant being of family Brassicaceae, preferably of genus Brassica, mostpreferably of genus Brassica napus, Brassica oleracea, Brassica nigra orBrassica carinata, to yield a F1 hybrid,ii) selfing the F1 hybrid for at least one generation, andiii) identifying the progeny of step (ii) comprising thepolynucleotides, construct or T-DNA capable of producing seed comprisingan increased tocopherol content as compared to seed of a control plant.

In an embodiment, said seed may comprise VLC-PUFA such that the contentof all VLC-PUFA downstream of 18:1 n-9 is at least 40% (w/w) of thetotal seed fatty acid content at an oil content of 40% (w/w), orpreferably the content of EPA is at least 8% (w/w) and/or the content ofDHA is at least 1% (w/w) of the total seed fatty acid content at an oilcontent of 30% (w/w), preferably at an oil content of 35% (w/w), andmore preferably at an oil content of 40% (w/w).

The method allows the creation of novel variants and transgenic speciesof plants of the present invention, and the seeds thereof. Such plantsand seeds exhibit the aforementioned benefits of the present invention.Preferably, the content of EPA is at least 10% by weight, even morepreferably at least 13% (w/w), of the total lipid content of the oil.Also preferably, the content of DHA is at least 1.5% by weight, evenmore preferably at least 2% (w/w), of the total lipid content of theoil. The present invention for the first time allows for the achievementof such high levels of tocopherol and VLC-PUFA in seed reliably underagronomic conditions, i.e. representative for the real yield obtainedfrom seeds of a commercial field of at least 1 ha planted with plants ofthe present invention, wherein the plants have a defined copy number ofgenes for implementing the pathway for production of EPA and/or DHA insaid plants, and the copy number being low, i.e. single-copy or partialdouble copy.

A plant of the present invention also includes plants obtainable orobtained by backcrossing (cross into the non-transgenic, isogenic parentline), and by crossing with other germplasms of the Triangle of U.Accordingly, the invention provides a method for creating a plant with agenotype that confers a heritable phenotype of an increased seed oiltocopherol content, obtainable or obtained from a progeny line preparedby a method comprising the steps of

i) crossing a transgenic plant of the invention (also called“non-recurring parent”) with a parent plant not expressing a genecomprised in the polynucleotides, T-DNA or construct of the presentinvention, said parent plant being of family Brassicaceae, preferably ofgenus Brassica, most preferably of genus Brassica napus, Brassicaoleracea, Brassica nigra or Brassica carinata, to yield a hybridprogeny,ii) crossing the hybrid progeny again with the parent to obtain anotherhybrid progeny,iii) optionally repeating step ii) andiv) selecting a hybrid progeny comprising the polynucleotides encodingdesaturases or elongases as set forth in the context of the method ofpresent invention, the T-DNA, or the construct of the present invention.

Backcrossing methods, e.g. as described above, can be used with thepresent invention to improve or introduce a characteristic into theplant line comprising the polynucleotides, construct or T-DNA of thepresent invention. Such hybrid progeny is selected in step iv) whichsuffices predetermined parameters. The backcrossing method of thepresent invention thereby beneficially facilitates a modification of thegenetic material of the recurrent parent with the desired gene, orpreferably the polynucleotides, construct, or T-DNA of the presentinvention, from the non-recurrent parent, while retaining essentiallyall of the rest of the desired genetic material of the recurrent parent,and therefore the desired physiological and morphological, constitutionof the parent line. The selected hybrid progeny is then preferablymultiplied and constitutes a line as described herein. Selection ofuseful progeny for repetition of step ii) can be further facilitated bythe use of genomic markers. For example, such progeny is selected forthe repetition of step ii) which comprises, compared to other progenyobtained in the previous crossing step, most markers also found in theparent and/or least markers also found in the non-recurring parentexcept the desired polynucleotides, construct, or T-DNA of the presentinvention or part of the T-DNA or construct thereof.

Preferably, a hybrid progeny is selected which comprises thepolynucleotides, construct or T-DNA of the present invention, and evenmore preferably also comprises at least one further expression cassettefrom the non-recurring parent of the present invention, e.g. byincorporation of an additional part of the construct or T-DNA of thepresent invention into the hybrid plant genetic material.

Further preferably a hybrid progeny is obtained wherein essentially allof the desired morphological and physiological characteristics of theparent are recovered in the converted plant, in addition to geneticmaterial from the non-recurrent parent as determined at the 5%significance level when grown under the same environmental conditions.

Further preferably, a hybrid progeny is selected which produces seedcomprising an increased tocopherol content as compared to a control, inparticular in the oil of seeds. Also preferably, the seed compriseVLC-PUFA such that the content of all VLC-PUFA downstream of 18:1n-9 isat least 40% (w/w) of the total seed fatty acid content at an oilcontent of 40% (w/w), or preferably the content of EPA is at least 8%(w/w) and/or the content of DHA is at least 1% (w/w) of the total seedfatty acid content at an oil content of 30% (w/w), preferably at an oilcontent of 35% (w/w), and more preferably at an oil content of 40%(w/w).

It is to be understood that such seed VLC-PUFA or tocopherol content isto be measured not from a single seed or from the seeds of an individualplant, but refers to the numeric average of seed VLC-PUFA content of atleast 100 plants, even more preferably of at least 200 plants, even morepreferably of at least 200 plants half of which have been grown in fieldtrials in different years.

The choice of the particular non-recurrent parent will depend on thepurpose of the backcross. One of the major purposes is to add somecommercially desirable, agronomically important trait to the line.

The term “line” refers to a group of plants that displays very littleoverall variation among individuals sharing that designation. A “line”generally refers to a group of plants that display little or no geneticvariation between individuals for at least one trait. A “DH (doubledhaploid) line,” as used in this application refers to a group of plantsgenerated by culturing a haploid tissue and then doubling the chromosomecontent without accompanying cell division, to yield a plant with thediploid number of chromosomes where each chromosome pair is comprised oftwo duplicated chromosomes. Therefore, a DH line normally displayslittle or no genetic variation between individuals for traits. Linescomprising one or more genes originally comprised in a T-DNA of thepresent invention in the non-recurring parent also constitute plants ofthe present invention.

The invention is also concerned with a method of plant oil and/ortocopherol production (in particular for tocopherol production),comprising the steps of

i) growing a plant of the present invention such as to obtainoil-containing seeds thereof,ii) harvesting said seeds, andiii) extracting oil from said seeds harvested in step ii).

Preferably the oil has an increased tocopherol content, in particular ascompared to the oil extracted from seeds of a control plant. Preferredincreased tocopherol contents are disclosed elsewhere herein.

The extraction step under iii) is preferably carried out underconditions which maintain the tocopherol content of the oil. Conditionswhich maintain the tocopherol content of the oil in the context of thepresent invention shall be conditions which do not reduce the tocopherolcontent. Such conditions are well known in the art and are e.g.described in Willner et al. Einflulß der Prozeßparameter auf dieTocopherolbilanz bei der Gewinnung von pflanzlichen Ölen. Lipid/Fett,Volume 99, Issue 4, pages 138-147, 1997 which herewith is incorporatedby reference in its entirety.

In addition, the oil may have a DHA content of at least 1% by weightbased on the total lipid content and/or an EPA content of at least 8% byweight based on the total lipid content.

In a further step, the method may comprise the step iv) of isolatingtocopherol from the oil extracted in step iii).

In an embodiment, the term “isolating tocopherol” means “enrichingtocopherol”.

How to isolate tocopherol from oil is well known in the art and e.g.described in “Commercial Extraction of Vitamin E from Food Sources” inThe Encyclopedia of Vitamin E, Preedy, V. R. and Watson R. R. (eds.),CABI Publishers, Oxford, U.K., pp. 140-152 and in U.S. Pat. No.5,627,289. Both documents are incorporated herein in their entirety.

For example, tocopherols can be isolated from by various methods such asesterification of the free fatty acids in the oil, by saponificationwhich allows for removal of fatty components from the oil, distillation,by chromatographic methods, by enzymatic methods (by using lipase) etc.These and further methods are described in the chapter of “TheEncyclopedia of Vitamin E” referred to in the previous paragraph indetail.

In an embodiment, the isolation comprises esterifying free fatty acidsin said oil with methanol; transesterifying triglycerides in said oil byalkali-catalyzed transesterification with methanol; acidifying and thenwashing the oil resulting from said transesterification; and removing bydistillation fatty acid methyl esters from the oil resulting from saidacidifying and washing. In an embodiment, steam distillates of the oilare used as the oil.

In an embodiment, an inorganic acid such as hydrochloric acid is usedfor the acidifying.

In an embodiment, 1 to 1.5 parts by volume of said mixture is esterifiedusing 1 part by volume of methanol.

In an embodiment, the free fatty acids are esterified at a temperatureof 60 to 100° C. (in particular at temperature of 65 to 70° C.).Preferably, the fatty acids are esterified the presence of a stronglyacidic ion exchanger.

Preferably, the oil comprises EPA, DHA, and/or DPA n-3 in concentrationsdescribed herein below.

Also preferably, the content of EPA is at least 8% by weight, even morepreferably at least 10% (w/w), of the total lipid content of the oil.Preferably, the content of DHA is at least 1% by weight, even morepreferably at least 1.5% (w/w), of the total lipid content of the oil.As described herein, the plant of the present invention comprises, forthe purposes of such method of plant oil production, preferablycomprises the polynucleotides, the construct, or the T-DNA of thepresent invention and optionally also one or more additional parts ofthe T-DNA or of the construct, wherein the part or parts, respectively,comprise at least one expression cassette of the T-DNA of the presentinvention.

The present invention also relates to oil comprising an increasedtocopherol content. Preferably, said oil is obtainable by theaforementioned methods, or produced by the plant of the presentinvention. Preferably, said oil also comprises an increased content ofVLC-PUFA (The term “high content” and “increased content” are usedinterchangeably herein). For example, the oil can comprise EPA, DHA,and/or DPA n-3 in concentrations described herein below.

The term “oil” refers to a fatty acid mixture comprising unsaturatedand/or saturated fatty acids which are esterified to triglycerides.Preferably, the triglycerides in the oil of the invention comprise PUFAor VLC-PUFA moieties as referred to above. The amount of esterified PUFAand/or VLC-PUFA is, preferably, approximately 30%, a content of 50% ismore preferred, a content of 60%, 70%, 80% or more is even morepreferred. The oil may further comprise free fatty acids, preferably,the PUFA and VLC-PUFA referred to above. For the analysis, the fattyacid content can be, e.g., determined by GC analysis after convertingthe fatty acids into the methyl esters by transesterification. Thecontent of the various fatty acids in the oil or fat can vary, inparticular depending on the source. The oil, however, shall have anon-naturally occurring composition with respect to the PUFA and/orVLC-PUFA composition and content. It is known that most of the fattyacids in plant oil are esterified in triacylglycerides. Accordingly, inthe oil of the invention, the PUFAs and VLC-PUFAs, preferably, alsooccur in esterified form in the triacylglcerides. It will be understoodthat such a unique oil composition and the unique esterification patternof PUFA and VLC-PUFA in the triglycerides of the oil shall only beobtainable by applying the methods of the present invention specifiedabove. Moreover, the oil of the invention may comprise other molecularspecies as well. Specifically, it may comprise minor amounts of thepolynucleotide or vector of the invention. Such low amounts, however,can be detected only by highly sensitive techniques such as PCR.

As described above, these oils, lipids or fatty acids compositions,preferably, comprise (by weight) 6 to 15% of palmitic acid, 1 to 6% ofstearic acid, 7-85% of oleic acid, 0.5 to 8% of vaccenic acid, 0.1 to 1%of arachic acid, 7 to 25% of saturated fatty acids, 8 to 85% ofmonounsaturated fatty acids and 60 to 85% of polyunsaturated fattyacids, in each case based on 100% and on the total fatty acid content ofthe organisms (preferably by weight). Preferred VLC-PUFAs present in thefatty acid esters or fatty acid mixtures is, preferably, 1% to 20% DHA,or 5.5% to 20% of DHA and/or 9.5% to 30% EPA based on the total fattyacid content (preferably by weight).

The oils, lipids or fatty acids according to the invention, preferably,comprise at least 1%, 2%, 3%, 4% 5.5%, 6%, 7% or 7.5%, more preferably,at least 8%, 9%, 10%, 11% or 12%, and most preferably at least 13%, 14%,15%, 16%. 17%, 18%, 19% or 20% of DHA, and/or at least 9.5%, 10%, 11% or12%, more preferably, at least 13%, 14%, 14.5%, 15% or 16%, and mostpreferably at least 17%, 18%, 19%, 20%. 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29% or 30% of EPA (preferably by weight) based on the totalfatty acid content of the production host cell, organism, advantageouslyof a plant, especially of an oil crop such as soybean, oilseed rape,coconut, oil palm, safflower, flax, hemp, castor-oil plant, Calendula,peanut, cacao bean, sunflower or the abovementioned othermonocotyledonous or dicotyledonous oil crops.

The seeds of the present invention shall comprise the oil or lipid ofthe present invention. Preferably, the oil or lipid is extracted,obtained, obtainable or produced from a plant, more preferably fromseeds of a plant or plants (in particular a plant or plants of thepresent invention). The oil or lipid thus can be obtained by the methodsof the present invention. In particular, the plant oil or plant lipid isan extracted plant oil or lipid. Also preferably, said oil or lipid isextracted, obtained, obtainable or produced from a plant, morepreferably from batches of seeds or bulked seeds of a plant or plants(in particular a plant or plants of the present invention).

Preferably, the term “extracted” in connection with an oil or lipidrefers to an oil or lipid that has been extracted from a plant, inparticular from seeds of a plant or plants. More preferably, the term“extracted” in connection with an oil or lipid refers to an oil or lipidthat has been extracted from a plant, in particular from batch of seedsor bulked seeds of a plant or plants. Such oil or lipid can be a crudecomposition. However, it may be also a purified oil or lipid in whiche.g. the water has been removed. In an embodiment, the oil or lipid isnot blended with fatty acids from other sources.

The oil or lipid of the present invention may be also an oil or lipid ina seed of plant. Preferably, said plant is a transgenic plant. Morepreferably, said plant is a plant of the present invention. In aparticular preferred embodiment, the plant is a Brassica plant.

The oil or lipid of the present invention shall comprise fatty acids. Inparticular, the oil or lipid shall comprise fatty acids in esterifiedform. Thus, the fatty acids shall be esterified. Preferably, the oil orlipid of the present comprises one or more of following fatty acids (inesterified form): Eicosapentaenoic acid (Timnodonic acid, EPA, 20:5n-3),Clupanodonic acid (DPA n-3), and DHA((Z,Z,Z,Z,Z,Z)-4,7,10,13,16,19-Docosahexaenoic acid). In an embodiment,the oil or lipid comprises EPA and DHA. Further, it is envisaged thatthe oil or lipid comprises EPA, DHA, and DPA n-3.

Preferred contents of the aforementioned fatty acids in the total fattyacid content of the lipid or oil of the present invention is furtherdescribed in the following. In the following, ranges are given for thecontents. The contents (levels) of fatty acids given herein areexpressed as percentage (weight of a particular fatty acid) of the totalweight of all fatty acids (present in the oil or lipid). The contentsare thus, preferably given as weight percentage (% w/w). The contentsgiven below are considered as high contents.

Preferably, the fatty acids are present in esterified form. Thus, thefatty acids shall be esterified fatty acids.

As set forth above, the oil or lipid may comprise EPA (20:5n-3).Preferably, the content of Eicosapentaenoic acid (Timnodonic acid, EPA,20:5n-3) is between 0.1% and 20%, more preferably between 2% and 15%,most preferably between 5% and 10% of the total fatty acid content.Further, it is envisaged that the content of EPA is between 5% and 15%of the total fatty acid content.

As set forth above, the oil or lipid may comprise Clupanodonic acid (DPAn-3). Preferably, the content of Clupanodonic acid (DPA n-3) is between0.1% and 10%, more preferably between 1% and 6%, most preferably between2% and 4% of the total fatty acid content. In addition, the content ofDPA n-3 may be at least 2% of the total fatty acids.

As set forth above, the oil or lipid may comprise DHA. Preferably, thecontent of DHA is between 1% and 10%, more preferably between 1% and 4%,most preferably between 1% and 2% of the total fatty acid content.Further, it is envisaged that the content of DHA is between 1% and 3% ofthe total fatty acid content.

A further embodiment according to the invention is the use of the oil,lipid, fatty acids and/or the fatty acid composition in feedstuffs,foodstuffs, dietary supplies, cosmetics or pharmaceutical compositionsas set forth in detail below. The oils, lipids, fatty acids or fattyacid mixtures according to the invention can be used for mixing withother oils, lipids, fatty acids or fatty acid mixtures of animal originsuch as, for example, fish oils.

The term “composition” refers to any composition formulated in solid,liquid or gaseous form. Said composition comprises the compound of theinvention optionally together with suitable auxiliary compounds such asdiluents or carriers or further ingredients. In this context, it isdistinguished for the present invention between auxiliary compounds,i.e. compounds which do not contribute to the effects elicited by thecompounds of the present invention upon application of the compositionfor its desired purpose, and further ingredients, i.e. compounds whichcontribute a further effect or modulate the effect of the compounds ofthe present invention. Suitable diluents and/or carriers depend on thepurpose for which the composition is to be used and the otheringredients. The person skilled in the art can determine such suitablediluents and/or carriers without further ado. Examples of suitablecarriers and/or diluents are well known in the art and include salinesolutions such as buffers, water, emulsions, such as oil/wateremulsions, various types of wetting agents, etc.

In a more preferred embodiment of the oil-, fatty acid orlipid-containing composition, the said composition is further formulatedas a pharmaceutical composition, a cosmetic composition, a foodstuff, afeedstuff, preferably, fish feed or a dietary supply.

The term “pharmaceutical composition” as used herein comprises thecompounds of the present invention and optionally one or morepharmaceutically acceptable carrier. The compounds of the presentinvention can be formulated as pharmaceutically acceptable salts.Acceptable salts comprise acetate, methylester, Hel, sulfate, chlorideand the like. The pharmaceutical compositions are, preferably,administered topically or systemically. Suitable routes ofadministration conventionally used for drug administration are oral,intravenous, or parenteral administration as well as inhalation.However, depending on the nature and mode of action of a compound, thepharmaceutical compositions may be administered by other routes as well.For example, polynucleotide compounds may be administered in a genetherapy approach by using viral vectors or viruses or liposomes.

Moreover, the compounds can be administered in combination with otherdrugs either in a common pharmaceutical composition or as separatedpharmaceutical compositions wherein said separated pharmaceuticalcompositions may be provided in form of a kit of parts. The compoundsare, preferably, administered in conventional dosage forms prepared bycombining the drugs with standard pharmaceutical carriers according toconventional procedures. These procedures may involve mixing,granulating and compressing or dissolving the ingredients as appropriateto the desired preparation. It will be appreciated that the form andcharacter of the pharmaceutically acceptable carrier or diluent isdictated by the amount of active ingredient with which it is to becombined, the route of administration and other well-known variables.The carrier(s) must be acceptable in the sense of being compatible withthe other ingredients of the formulation and being not deleterious tothe recipient thereof. The pharmaceutical carrier employed may be, forexample, a solid, a gel or a liquid. Exemplary of solid carriers arelactose, terra alba, sucrose, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid and the like. Exemplary of liquidcarriers are phosphate buffered saline solution, syrup, oil such aspeanut oil and olive oil, water, emulsions, various types of wettingagents, sterile solutions and the like. Similarly, the carrier ordiluent may include time delay material well known to the art, such asglyceryl mono-stearate or glyceryl distearate alone or with a wax. Saidsuitable carriers comprise those mentioned above and others well knownin the art, see, e.g., Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa. The diluent(s) is/are selected so as notto affect the biological activity of the combination. Examples of suchdiluents are distilled water, physiological saline, Ringer's solutions,dextrose solution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation may also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. Atherapeutically effective dose refers to an amount of the compounds tobe used in a pharmaceutical composition of the present invention whichprevents, ameliorates or treats the symptoms accompanying a disease orcondition referred to in this specification.

The term “cosmetic composition” relates to a composition which can beformulated as described for a pharmaceutical composition above. For acosmetic composition, likewise, it is envisaged that the compounds ofthe present invention are also, preferably, used in substantially pureform. Impurities, however, may be less critical than for apharmaceutical composition. Cosmetic compositions are, preferably, to beapplied topically.

Preferred cosmetic compositions comprising the compounds of the presentinvention can be formulated as a hair tonic, a hair restorercomposition, a shampoo, a powder, a jelly, a hair rinse, an ointment, ahair lotion, a paste, a hair cream, a hair spray and/or a hair aerosol.

Seeds of three events described in detail in the examples section belowhave been deposited at ATCC under the provisions of the Budapest treatyon the International Recognition of the Deposit of Microorganisms forthe Purposes of Patent Procedure, i.e. seeds of event “LBFLFK”=ATCCDesignation “PTA-121703”, seeds of event “LBFDHG”=ATCC designation“PTA-121704”, and seeds of the event “LBFDAU”=ATCC Designation“PTA-122340”. Applicants have no authority to waive any restrictionsimposed by law on the transfer of biological material or itstransportation in commerce. Applicants do not waive any infringement oftheir rights granted under this patent or rights applicable to thedeposited events under the Plant Variety Protection Act (7 USC sec.2321, et seq.), Unauthorized seed multiplication prohibited. This seedmay be regulated according to national law. The deposition of seeds wasmade only for convenience of the person skilled in the art and does notconstitute or imply any confession, admission, declaration or assertionthat deposited seed are required to fully describe the invention, tofully enable the invention or for carrying out the invention or any partor aspect thereof. Also, the deposition of seeds does not constitute orimply any recommendation to limit the application of any method of thepresent invention to the application of such seed or any materialcomprised in such seed, e.g. nucleic acids, proteins or any fragment ofsuch nucleic acid or protein.

The deposited seeds are derived from plants that were transformed withthe T-DNA vector having a sequence as shown in SEQ ID NO: 3.

The invention is further described by means of accompanying examples,which, however, are not intended to limit the scope of the inventiondescribed herein.

EXAMPLES Example 1: Materials and Methods A. General Cloning Methods

Cloning methods as e.g. use of restriction endonucleases to cut doublestranded DNA at specific sites, agarose gel electrophoreses,purification of DNA fragments, transfer of nucleic acids ontonitrocellulose and nylon membranes, joining of DNA-fragments,transformation of E. coli cells and culture of bacteria were performedas described in Sambrook et al. (1989) (Cold Spring Harbor LaboratoryPress: ISBN 0-87965-309-6). Polymerase chain reaction was performedusing Phusion™ High-Fidelity DNA Polymerase (NEB, Frankfurt, Germany)according to the manufacturer's instructions. In general, primers usedin PCR were designed such that at least 20 nucleotides of the 3′ end ofthe primer anneal perfectly with the template to amplify. Restrictionsites were added by attaching the corresponding nucleotides of therecognition sites to the 5′ end of the primer. Fusion PCR, for exampledescribed by K. Heckman and L. R. Pease, Nature Protocols (2207) 2,924-932 was used as an alternative method to join two fragments ofinterest, e.g. a promoter to a gene or a gene to a terminator. GeneSynthesis, as for example described by Czar et al. (Trends inBiotechnology, 2009, 27(2): 63-72), was performed by Life Technologiesusing their Geneart® service. The Geneart® technology, described inWO2013049227 allows production of genetic elements of a few basepair(bp) in length, and was used in this invention to produce entireplasmids of about 60,000 bp. Chemical synthesis of nucleotides topolynucleotides was employed for short DNA fragments, which were thencombined in a sequential, modular fashion to fragments of increasingsize using a combination of conventional cloning techniques as describedin WO2013049227.

B. Different Types of Plant Transformation Plasmids Suitable to Transferof Multiple Expression Cassettes Encoding Multiple Proteins into thePlant Genome.

For agrobacteria based plant transformation, DNA constructs preferablymeet a number of criteria: (1) The construct carries a number of geneticelements that are intended to be inserted into the plant genome on a socalled Transfer DNA (T-DNA) between a ‘T-DNA Left Border’ (LB) and‘T-DNA Right Border’ (2) The construct replicates in E. coli, becausemost cloning steps require DNA multiplication steps in E. coli. (3) Theconstruct replicates in Agrobacterium (e.g. A. tumefaciens or A.rhizogenes), because the plant transformation methods rely on usingAgrobacterium to insert the genetic elements of interest into the plantgenome of a cell that was infected by Agrobacterium. (4) The constructcontains supporting genetic elements that encode proteins which arerequired for infection of the plant cell, and for transfer andintegration of desired genetic elements into the plant genome of anplant cell infected by the Agrobacterium, or the construct was used incombination with a second construct containing such supporting geneticelements that was present in the same Agrobacterium cell. (5) Theconstructs can contain selection markers to facilitate selection oridentification of bacterial cells that contain the entire construct, andof a plant cell(s) that contains the desired genetic elements. Anoverview of available plasmids was given in Komori et al (2007).

C. Assembly of Genes Required for EPA and DHA Synthesis within BiBACT-Plasmids Containing the F Factor/pRI Origin of Replication

For synthesis of VLC-PUFA in Brassica napus seeds, the set of genesencoding the proteins of the metabolic VLC-PUFA pathway were combinedwith expression elements (promoter, terminators and introns) andtransferred into a binary t-plasmid that was used for agrobacteriamediated transformation of plants. All expression cassettes have beencombined onto a single binary T-plasmid. The advance of DNA synthesisallows numerous companies to offer services to use a combination ofchemical synthesis and molecular biological techniques to synthesize denovo, without an initial template, polynucleotides up to the size ofmicrobial genomes. Synthesis used in the construction of the plasmiddescribed in this example was performed by Life Technologies using theirGeneart® service. The Geneart® technology, described in WO2013049227allows production of genetic elements of a few basepair (bp) length, andwas used in this invention to produce the binary T-plasmid for planttransformation VC-LTM593-1qcz rc having a total size of ˜61.000 bp. Thestructure of the plasmid VC-LTM593-1qcz rc is given in Table 1.

TABLE 1 Genetic Elements of plasmid VC-LTM593-1qcz rc. Listed are thenames of the elements, the position in VC-LTM593-1qcz rc (nucleotidenumber, note: start position was larger than stop position for elementsencoded by the complementary strand of VC-LTM593-1qcz rc), the functionand source of the element. The T-DNA integrated into the plant genomeduring the transformation process was flanked by a right border(nucleotides 59895 to 148 of VC-LTM593- 1qcz rc) and a left border(nucleotides 43830 to 43695 of VC-LTM593-1qcz rc). Elements outside ofthat region (=vector backbone) are required for cloning and stablemaintenance in E. coli and/or agrobacteria. The sequence of this vectoris shown in SEQ ID NO: 3. The locations (of the e.g. of promoters,genes, introns, terminators and separators) in SEQ ID NO: 3 are indictedin the second and third column. Genetic Elements of plasmid VC-Description, Function and Source of LTM593-1qcz rc From To Elementp-VfUSP_684bp[LLL894] 329 1012 Promoter from UNKNOWN SEED PROTEIN geneUSP (accession: X56240) from Vicia faba i-Atss18_252[LJK36] 1013 1264i-Atss18_252bp functional intron region; intron with partial 5′ UTR,Arabidopsis thaliana, Locus At1g01170, +37 to +288 bp (numberingrelative to start of transcription) (+72 to +282 bp 5′UTR-Intron only)c-d6Elo(Pp_GA2) 1267 2139 Delta-6 ELONGASE from Physcomitrella patenst-CaMV35S 2140 2355 Terminator CaMV35S from 35S gene from Cauliflowermosaic virus p-LuCnl(1064bp) 2448 3511 Promoter from CONLININ gene fromLinum usitatissimum i-Atss14_377bp[LJK32] 3512 3888i-Atss14_377bp[LJK32] functional intron region; intron with partial5′UTR, Arabidopsis thaliana, Locus At5g63190, +166 to +542 bp (numberingrelative to start of transcription) (+201 to +542 bp 5′UTR-Intron only)c-d5Des(Tc_GA2) 3892 5211 Delta-5 DESATURASE from Thraustochytrium sp.ATCC21685 t-AgrOCS 192bp[LED12] 5212 5403 Terminator from OCTOPINESYNTHASE gene OCS from Agrobacterium tumefaciens p-SBP 5539 7337Promoter from a SUCROSE- BINDING PROTEIN-RELATED gene from Vicia fabai-Atss2_455bp[LJK20] 7338 7792 i-Atss2_455bp functional intron region;intron with partial 5′UTR, Arabidopsis thaliana, Locus At1g65090, +77 to+531 bp (numbering relative to start of transcription) (+113 to +508 bp5′UTR-Intron only) c-d6Des(Ot_febit) 7802 9172 Delta-6 DESATURASE fromOstreococcus tauri t-StCATHD-pA 9200 9434 Terminator from CATHEPSIN DINHIBITOR gene [CATHD] from Solanum tuberosum [Potato] p-LuPXR1727bp[LLL823] 9513 11239 Promoter from PEROXIREDOXIN LIKE protein genePXR from Linum usitatissimum i-Atss1_846bp[ltm593] 11240 12085i-Atss1_847bp functional intron region; intron with partial 5′ UTR,Arabidopsis thaliana, Locus At1g62290 (aspartyl protease familyprotein), +1 to +847 bp (numbering relative to start of transcription)(+19 to +841 bp 5′UTR-Intron only); 1 bp at poly T stretch shortercompared to original i-Atss1_847bp c-d6Elo(Tp_GA2) 12099 12917 Delta-6ELONGASE from Thalassiosira pseudonana t-AtPXR 400bp[LLL823] 12973 13372Terminator from peroxiredoxin like protein gene PXR (At1g48130) fromArabidopsis thaliana p-Napin A/B 13542 14205 Promoter from napA/B gene(napin, seed storage protein) from Brassica napus i-Atss14_377bp[LJK32]14206 14582 i-Atss14_377bp[LJK32] functional intron region; intron withpartial 5′ UTR, Arabidopsis thaliana, Locus At5g63190, +166 to +542 bp(numbering relative to start of transcription) (+201 to +542 bp5′UTR-Intron only) c-d12Des(Ps_GA2) 14589 15785 Delta-12 DESATURASE fromPhythophthora sojae t-E9 15804 16361 Terminator from Small Subunit ofRuBisCo rbcS gene (E9) from Pisum sativum p-BnSETL-v1[1234bp] 1645417687 SETL-v1 Brassica napus promoter c-o3Des(Pir_GA) 17690 18781Omega-3 DESATURASE from Pythium irregulare t-BnSETL 18803 19416 SETL-v1Brassica napus terminator p-VfUSP_684bp[LLL894] 19495 20178 Promoterfrom UNKNOWN SEED PROTEIN gene USP (accession: X56240) from Vicia fabai-Atss18_252[LJK36] 20179 20430 i-Atss18_252bp functional intron region;intron with partial 5′ UTR, Arabidopsis thaliana, Locus At1g01170, +37to +288 bp (numbering relative to start of transcription) (+72 to +282bp 5′UTR-Intron only) c-o3Des(Pi_GA2) 20441 21526 Omega-3-DESATURASEfrom Phythophthora infestans t-CaMV35S 21535 21750 Terminator CaMV35Sfrom 35S gene from Cauliflower mosaic virus p-BnSETL-v1[1234bp] 2188623119 SETL-v1 Brassica napus promoter c-d5Des(Tc_GA2) 23122 24441Deita-5 DESATURASE from Thraustochytrium sp. ATCC21685 t-BnSETL 2446325076 SETL-v1 Brassica napus terminator p-ARC5_perm1 25223 26373Promoter derived from a promoter from ARCILINE 5 gene from Phaseolusvulgaris c-d4Des(Tc_GA3) 26384 27943 Delta-4 DESATURASE fromThraustochytrium sp. t-pvarc 27957 28556 Terminator of ARC5 gene fromPhaseolus vulgaris p-LuPXR 1727bp[LLL823] 28649 30375 Promoter fromPEROXIREDOXIN LIKE protein gene PXR from Linum usitatissimumi-Atss15_758bp[LJK33] 30376 31133 i-Atss15_758bp[LJK33] functionalintron region; intron with partial 5′UTR, Arabidopsis thaliana, LocusAt2g27040, +93 bp to +850 bp (numbering relative to start oftranscription) (+128 to +847 bp 5′UTR-Intron only) c-o3Des(Pir_GA) 3114932240 Omega-3 DESATURASE from Pythium irregulare t-AtPXR 400bp[LLL823]32297 32696 Terminator from PEROXIREDOXIN LIKE protein gene PXR(At1g48130) from Arabidopsis thaliana p-LuCnl(1064bp) 32832 33895Promoter from CONLININ gene from Linum usitatissimumi-Atss2_455bp[LJK20] 33896 34350 i-Atss2_455bp functional intron region;intron with partial 5′UTR, Arabidopsis thaliana, Locus At1g65090, +77 to+531 bp (numbering relative to start of transcription) (+113 to +508 bp5′UTR-Intron only) c-d4Des(PI_GA)2 34360 35697 Delta-4 DESATURASE fromPavlova lutheri t-AgrOCS 192bp[LED12] 35719 35910 Terminator fromOCTOPINE SYNTHASE gene OCS from Agrobacterium tumefaciens p-BnFae1 3610437533 Promoter from Beta-KETOACYL-CoA SYNTHASE (FAE1.1) gene fromBrassica napus i-Atss1_847bp[LJK19] 37534 38380 i-Atss1_847bp functionalintron region; intron with partial 5′ UTR, Arabidopsis thaliana, LocusAt1g62290 (aspartyl protease family protein), +1 to +847 bp (numberingrelative to start of transcription) (+19 to +841 bp 5′UTR-Intron only);from QC1153-1/RTP6393. c-d5Elo(Ot_GA3) 38388 39290 Delta-5 ELONGASE fromOstreococcus tauri t-bnFae1 39307 39706 Terminator from FATTY ACIDELONGASE (FAE1, At4g34520) gene of Arabidopsis thalianap-YPC105906_PcUbi4-2[long] 39830 40806 MTX Parsley UBI4-2 promoter withinternal intron c- 40814 42826 ACETOHYDROXYACID SYNTHASEAtAHASL_A122T_S653N[minusRES] LARGE-SUBUNIT gene/CDS from Arabidopsiswith S653N (csr1-2) mutation and A122T SDM mutation minus restrictionsites t-AtAHAS-3′UTR[rtp4820] 42827 43606 Arabidopsis (dicot) AtAHASL 3′Un- translated Region [trimmed] terminator for ACETOHYDROXYACID SYNTHASEgene b-LLB 43830 43695 Left T-DNA Left border from pTi15955 [Genbank#AF242881] c-KanR_Tn903 45777 44962 Kanamycin Resistance selectiongene/CDS p-Kan[lm500] 45898 45778 Promoter for Kanamycin resistance geneo-ori-2 47051 47267 ori-2 origin of replication c-repE 47361 48116 repEgene/CDS c-sopA 48695 49870 sapA gene/CDS c-sopB 49870 50841 sopBgene/CDS c-sopC/incD 50914 51387 incDlsopC partial gene/CDS c-tral 5189051949 trat gene/CDS mf-tral - repA intergenic region 51938 52300regulatory region of traR dependent quorum sensing regulon - containing2 tra-boxes (see LI AND FARRAND JOURNAL OF BACTERIOLOGY, January 2000,p. 179-188) o-repA 52301 53518 Rep-A gene from pTiC58 replicon (LI ANDFARRAND JOURNAL OF BACTERIOLOGY, January 2000, p. 179 . . . 188) rr-repB53748 54758 rep-B gene from pTiC58 replicon (LI AND FARRAND JOURNAL OFBACTERIOLOGY, January 2000, p. 179 . . . 188) o-repC 54973 56292 rep-Cgene from pTiC58 replicon (LI AND FARRAND JOURNAL OF BACTERIOLOGY,January 2000, p. 179 . . . 188) mf-y4cG 56771 56301 fragment of DNAinvertase homolog; similar to Rhizobium sp. NGR234 pNGR234a Y4CG tr-Tn558811 57250 Transposon Tn5 sequence o-oriT 59107 59275 oriT from pRK310genbank file b-RB[rtp4394] 148 59895 Right T-DNA Right border

D. Procedure for Production of Transgenic Plants Using BiBACs

In general, the transgenic rapeseed plants were generated by a modifiedprotocol according to DeBlock et al. 1989, Plant Physiology,91:694-701). Overnight cultures of the strain intended to be transformedwas prepared in YEB medium with antibiotics (20 mg/L chloramphenicol, 5mg/L tetracycline, 50 mg/L kanamycin) and grown at 28° C. On the nextday the optical density of the culture was checked at 600 nm wavelength. It reached about 1.0. Cultures of lower optical density wereextended in cultivation period. Cultures with an optical density ofabove 1.3 were diluted with YEB medium to an OD of approximately 0.2 andcultured until they reached an OD of 1.0. Cultures were pelleted atabout 4000 g and re-suspended in liquid MS medium (Murashige and Skoog1962), pH 5.8, 3% sucrose with 100 mg/L Acetosyringone to reach an OD₆₀₀nm of 0.1. The Agrobacterium suspensions were used for inoculation ofhypocotyl segments prepared from 5 days old etiolated seedlings.

Seeds were germinated for five days under low light conditions (<50μMol/m2s) using MSB5 medium from Duchefa (Duchefa Biochemie, PO Box 8092003 R V Haarlem, Netherlands), pH 5.8, 3% sucrose and 0.8% Oxoid agar.Germination under light conditions produces explants, which are morestable and easier to handle compared to etiolated hypocotyls. Hypocotylsegments of 4 to 7 mm length were inoculated in a bath of Agrobacteriumcells under gentle shaking up to 4 min and sieved after the incubation.Infected explants were transferred to petri dishes with co-cultivationmedium (MS medium, pH 5.6, 3% sucrose, 0.6 g/L MES(2-(N-Morpholino)ethanesulfonic acid), 18 g/L mannitol, 0.7% phytoagar(Duchefa Biochemie, PO Box 809 2003 R V Haarlem, Netherlands, partnumber SKU:P1003), 100 mg/L Acetosyringone, 200 mg/L L-Cysteine, 1 mg/L2,4D (2,4-Dichlorophenoxyacetic acid)) carrying one layer of Whatmanfilter paper on its surface. Petri dishes were sealed with tape andincubated at 23 C under long day conditions (16 h light/8 h darkness)for three days. After the three days co-cultivation period explants weretransferred to MS medium, pH 5.6, 3% sucrose, 0.6 g/L MES, 18 g/Lmannitol, 07% Phytoagar, 1 mg/L 2,4D and 500 mg/L Carbenicillin toprevent Agrobacterium growth and incubated for a recovery period underthe same physical conditions as for the co-cultivation for 7 days.

For selective regeneration explants were transferred after the recoveryperiod to MS medium, pH 5.8, 3% sucrose, 0.7% Phytoagar, 2.5 mg/L AgNO₃,3 mg/L BAP (6-Benzylaminopurine), 0.1 mg/L GA (Gibberellic acid), 0.1mg/L NAA (1-Naphthaleneacetic acid), 500 mg/L Carbenicillin, 100 nMImazethapyr (Pursuit) and cultured for two weeks under long dayconditions as described above. Sub-cultivation takes place every twoweeks. Hormones were stepwise reduced as follows: BAP 3 to 0.5 to 0.05mg/L; GA (Gibberellic acid) 0.1 to 0.25 to 0.25 mg/L; NAA 0.1 to 0 to 0mg/L.

Developing shootlets could be harvested after the second cycle ofselective regeneration. Shootlets were cut and transferred to eitherElongation/rooting medium (MS medium, pH 5.8, 2% sucrose, 100 mg/Lmyo-inositol, 40 mg/L Adenine sulphate, 500 mg/L MES, 0.4% Sigma Agar,150 mg/L Timentin, 0.1 mg/L IBA (Indole-3-butyric acid)) or to rockwool/stone wool or foam mats (Grodan, GRODAN Group P.O. Box 1160, 6040KD Roermond The Netherlands, or Oasis, 919 Marvin Street, Kent, Ohio44240 USA) watered with 1/10 Vol. of MS medium, pH 5.8 without sucroseunder ex vitro long day conditions in covered boxes.

Shoots were elongated and rooted in in vitro medium and were transferreddirectly to soil. Either in vitro shoots or GH adapted shoots weresampled for molecular analysis.

Medium were used either autoclaved (except antibiotics, hormones,additives such as L-cysteine, Acetosyringon, imidazolinone components)or filter sterilized prepared (Agar component autoclaved, allowed tocool to 42 C and then used).

E. Seed Germination and Plant Growth in the Greenhouse and Field

Transformed plants were cultivated for seed production and phenotypicassessment in both the greenhouse and in the field. Greenhouse growthconditions were a sixteen hour light period followed by an eight hourdark period. The temperature was 20 degrees celsius during the lightperiod (also called the day period) with a level of light correspondingto 200-300 micromoles of photons m−2 s−1 (this is the incident of lightat the top of the plant and lights were adjusted in terms of distancefrom the plant to achieve this rate). During the day period the range oflight in the greenhouse varied between 130 and 500 micromoles of photonsm−2 s−1. Getting out of the day range just cited triggered either theuse of artificial light to bring the level up to 200-300 micromoles ofphotons m−2 s−1 or shading and/or shut off of lights to bring the levelback to 200-300 micromoles of photons m−2 s−1. The dark period (alsoreferred to as the night period) temperature was 18 C. Four hours beforethe light period began the temperature was lowered to 15 C for theremainder of the dark period. Plants were irrigated and treated forinsects as necessary. The soil type was 50% Floradur B Seed+50% FloradurB Cutting (including sand and perlite) provided by Floragard (Oldenburg,Germany). Plant growth was enhanced by nutrient supplementation.Nutrients were combined with the daily watering. A 0.1% (w/v) fertilizersolution (Hakaphos Blue 15(N)-10 (P)-15(K), Compo GmbH & Co KG, MOnster,Germany) was used to water the plants. Water was supplied on demand(e.g. depending on plant growth stage, water consumption etc.). To avoidcross-pollination, plants were bagged at the time when the first flowersopened. Plants were checked daily in order to ensure that all openflowers were covered by the bags. Open flowers that were not coveredproperly were removed.

For field grown plants, the plants were grown in six locations whichcorrespond climatically to USDA growth zones 3a-4b and 5a. The plantsgrown in the regions corresponding to USDA growth zones 3a-4b and 5awere grown in the summer. Standard horticultural practices for canolawere followed. Netting and other measures to protect from birds andinsects were used as deemed necessary by the growers, as were herbicidesand fertilizer applications. The planting density for all locations waseighty seeds per square meter.

F. Lipid Extraction and Lipid Analysis of Plant Oils

The results of genetic modifications in plants or on the production of adesired molecule, e.g. a certain fatty acid, were determined by growingthe plant under suitable conditions, e.g. as described above, andanalyzing the growth media and/or the cellular components for enhancedproduction of the desired molecule, e.g. lipids or a certain fatty acid.Lipids were extracted as described in the standard literature includingUllman, Encyclopedia of Industrial Chemistry, Bd. A2, S. 89-90 und S.443-613, VCH: Weinheim (1985); Fallon, A., et al., (1987) “Applicationsof HPLC in Biochemistry” in: Laboratory Techniques in Biochemistry andMolecular Biology, Bd. 17; Rehm et al. (1993) Biotechnology, Bd. 3,Kapitel III: “Product recovery and purification”, S. 469-714, VCH:Weinheim; Belter, P. A., et al. (1988) Bioseparations: downstreamprocessing for Biotechnology, John Wiley and Sons; Kennedy, J. F., undCabral, J. M. S. (1992) Recovery processes for biological Materials,John Wiley and Sons; Shaeiwitz, J. A., und Henry, J. D. (1988)Biochemical Separations, in: Ullmann's Encyclopedia of IndustrialChemistry, Bd. B3; Kapitel 11, S. 1-27, VCH: Weinheim; and Dechow, F. J.(1989) Separation and purification techniques in biotechnology, NoyesPublications.

It is acknowledged that extraction of lipids and fatty acids can becarried out using other protocols than those cited above, such asdescribed in Cahoon et al. (1999) Proc. Natl. Acad. Sci. USA 96(22):12935-12940, and Browse et al. (1986) Analytic Biochemistry152:141-145. The protocols used for quantitative and qualitativeanalysis of lipids or fatty acids are described in Christie, William W.,Advances in Lipid Methodology, Ayr/Scotland: Oily Press (Oily PressLipid Library; 2); Christie, William W., Gas Chromatography and Lipids.A Practical Guide—Ayr, Scotland: Oily Press, 1989, Repr. 1992, IX, 307S. (Oily Press Lipid Library; 1); “Progress in Lipid Research, Oxford:Pergamon Press, 1 (1952)-16 (1977) u.d.T.: Progress in the Chemistry ofFats and Other Lipids CODEN.

To generate transgenic plants containing the genetic elements describedin example 1C for production of EPA and DHA in seeds, rapeseed (Brassicanapus) was transformed as described in 1D. Selected plants containingthe genetic elements were grown until development of mature seeds underthe conditions cited in Example 1E. Fatty acids from harvested seedswere extracted as described above and analyzed using gas chromatographyas described above. The content (levels) of fatty acids is expressedthroughout the present invention as percentage (weight of a particularfatty acid) of the (total weight of all fatty acids) contained in theoil of seeds. Seed oil content is expressed throughout the presentinvention as percentage of (oil weight) of the (total oil weight ofseeds).

G. Compositional Analysis of Plant Seed Samples

The effect of genetic modification on seed composition was determined bygrowing plants under suitable conditions, e.g. as described above, andanalyzing seed tissue for specific compositional parameters. Mature seedsamples were milled into fine powder using a Foss Knifetec 1095 SampleMill and provided to Eurofins Nutrition Analysis Center (ENAC).Specifically, Vitamin E (tocopherol) content was measured by in milledseeds samples by ENAC using methods MET-VT-008 and MET-VT-030, both ofwhich refer to the Association Of Analytical Communities method AOAC971.30, and involve HPLC separation and quantification.

Example 2: Plants Containing the T-DNA of Plasmid VC-LTM593-1Qcz Rc forEnhanced Production of Tocopherol, and EPA and DHA in Seeds

All genetic elements described in this example were transferred on asingle T-DNA using a BiBAC plasmid into the plant genome. To this end,the plasmid VC-LTM593-1qcz rc was cloned into agrobacteria, and planttissue was incubated according to example 1 with this agrobacterialculture. The genetic elements of VC-LTM593-1qcz rc and the function ofeach element are listed in Table 1. For convenience, all enzymesexpressed in seeds of plants carrying both T-DNA of VC-LTM593-1qcz rcare additionally listed Table 2. In an embodiment, the plant, plant part(in particular seed), T-DNA, or construct of the present inventioncomprises some desaturases and/or elongases, or all desaturases and/orelongases as disclosed in the Table.

TABLE 2 List of genes carried by the T-DNA of plasmid VC-LTM593-1qcz rc.Preferred polynucleotide and protein sequences are shown in column 4 and5. Genes encoding Protein enzmyes for EPA Length Enzymatic function andPolynucleotide sequence and DHA synthesis (bp) source of encoded proteinSEQ ID NO: SEQ ID NO c-d12Des(Ps_GA2) 1197 Delta-12 desaturase 265 266from Phythophthora sojae c-d6Des(Ot_febit) 1371 Delta-6 desaturase 261262 from Ostreococcus tauri c-d6Elo(Pp_GA2) 873 Delta-6 elongase from257 258 Physcomitrella patens c-d6Elo(Tp_GA2) 819 Delta-6 elongase from263 264 Thalassiosira pseudonana 2 copies of c- 1320 Delta-5 desaturase259 260 d5Des(Tc_GA2) from Thraustochytrium sp. ATCC21685c-o3Des(Pi_GA2) 1086 Omega-3-desaturase 269 270 from Phythophthorainfestans 2 copies of c- 1092 Omega-3 desaturase 267 268 o3Des(Pir_GA)from Pythium irregulare c-d5Elo(Ot_GA3) 903 Delta-5 elongase from 275276 Ostreococcus tauri c-d4Des(Pl_GA)2 1338 Delta-4 desaturase 273 274from Pavlova lutheri c-d4Des(Tc_GA3) 1560 Delta-4 desaturase 271 272from Thraustochytrium sp.A. Fatty Acid Profiles, and Vitamin E Content of T2 Plants CarryingT-DNAs of Plasmids VC-LTM593-1Qcz Rc Cultivated in Field Trials in USDAGrowth Zones 3a-4b and 5a During the SummerHomozygous T2 Plants from Six Independent Transgenic Events thatContained 1-2 Copies of the T-DNA VC-LTM593-1qcz rc were grown in fieldlocations according example 1. The T3 seeds were harvested and submittedfor fatty acid analysis as described in example 1. Table 3 containsfatty acid profile data across all samples from all locations, for eachevent. Every event is capable of making VLC-PUFAs in the field (ARA, EPAand DHA).

The same T3 seeds described in Table 3 were submitted for compositionalanalysis as described in example 1. To analyze the data, ANOVA wasconducted using the software JMP 11.0. Analysis was conducted at the 95%confidence level using Tukey test. To compensate for unbalance in thedata obtained from the field trial (e.g. due to e.g. weather), LeastSquare means instead of means where used in the statistical analysis.Common letters in Table 3 indicate no significant difference of theleast square means. Based on this statistical analysis, one event,LBFDAU, contains higher gamma tocopherol and total tocopherol than theuntransformed Kumily control, while all other events tend to have highergamma- and total tocopherol levels than Kumily, with the exception ofevent LBFIHE.

The transgenic events described in Tables 3 and 4 all have decreased18:1+18:2 content relative to untransformed Kumily (18:1+18:2=80%).However, we did not observe any significant decrease in alpha-tocopherolcontent as would have been predicted based on Li et μL (2013) J AgricFood Chem 61:34-40. Instead, we observe increases in gamma-tocopheroland total tocopherol content, with the largest increase occurring inevent LBFDAU that produces the most combined EPA+DHA. A correlationanalysis was performed to reveal correlations between VLC-PUFA andtocopherols (Table 5). There we no significant correlations between ARA(an n-6 fatty acid) and any tocopherol components. On the other hand,significant positive correlations were observed between varioustocopherols and EPA and DHA. Correlations coefficients were determinedfor the sum of all n-3 or all n-6 fatty acids 20 carbons in length orgreater. The correlation between tocopherol of VLC-PUFA content isspecific to n-3 fatty acids. The highest correlations were observedbetween n-3 fatty acids 20 carbon in length or greater and gamma-,delta-, and total tocopherols. Therefore, introduction of a biosyntheticpathway that synthesizes the 20 and 22 carbon n-3 VLC-PUFAs EPA, DPA,and DHA into plants also results in an increase in vitamin E content.

TABLE 3 Fatty acid profiles of T3 seeds harvested from T2 plantscultivated in the field, corresponding to USDA growth zones 3a-4b and5a, for field trials of canola events containing the T-DNAs of plasmidVC-LTM593-1qcz rc. The events are indicated in the first column, alongwith the number of T3 seed aliquots representing a plot were measuredper event For event LBFGKN, 36 plots and 60 single plants from thoseplots where measured. Per seed batch a random selection of ~15 seed wasmeasured in five technical repeats. Values are the least square means ±standard deviation. 16:1 16:3 18:1 18:2 18:2 18:3 18:3 18:4 20:1 20:220:3 Event 16:0 n-7 n-3 18:0 n-9 n-6 n-9 n-3 n-6 n-3 20:0 n-9 n-6 n-3LBFDAU 4.7 ± 0.2 ± 0 ± 2.7 ± 28.6 ± 29.2 ± 1 ± 6.1 ± 1.6 ± 0.3 ± 0.7 ±0.7 ± 0.1 ± 0.1 ± (n = 16) 0.1 0 0 0.1 1.5 0.7 0.1 0.3 0.1 0 0 0 0 0LBFDGG 4.7 ± 0.2 ± 0 ± 2.5 ± 34.2 ± 32.3 ± 0.6 ± 7 ± 1.2 ± 0.2 ± 0.6 ±0.8 ± 0.1 ± 0.1 ± (n = 36) 0.1 0 0 0.2 1.9 1.2 0.1 0.5 0.1 0 0 0 0 0LBFGKN 4.6 ± 0.2 ± 0 ± 2.6 ± 33.7 ± 32.8 ± 0.6 ± 7.5± 0.9 ± 0.2 ± 0.7 ±0.8 ± 0.2 ± 0.1 ± (n = 36 + 60) 0.2 0 0 0.2 1.7 1.4 0.1 0.6 0.1 0 0 0.10 0 LBFIHE 4.8 ± 0.2 ± 0 ± 2.6 ± 31.2 ± 33.9 ± 0.6 ± 6.7 ± 1.3 ± 0.3 ±0.7 ± 0.8 ± 0.2 ± 0.1 ± (n = 36) 0.2 0 0 0.2 1.7 1.2 0.1 0.7 0.2 0 0.1 00 0 LBFLFK 4.7 ± 0.2 ± 0 ± 2.6 ± 30.1 ± 30.2 ± 0.9 ± 6.2 ± 1.5 ± 0.3 ±0.6 ± 0.8 ± 0.1 ± 0.1 ± (n = 36) 0.2 0 0 0.2 1.9 1.1 0.1 0.4 0.2 0.1 0 00 0 LBFPRA 4.8 ± 0.2 ± 0 ± 2.6 ± 28.4 ± 32.7 ± 0.8 ± 5.7 ± 1.6 ± 0.3 ±0.7 ± 0.8 ± 0.2 ± 0.1 ± (n = 36) 0.2 0 0 0.2 2.1 1.4 0.1 0.4 0.2 0.1 0 00 0 20:3 20:4 20:4 20:5 22:1 22:4 22:5 22:5 22:6 22:4 20:2 Event n-6 n-3n-6 n-3 22:0 n-9 n-6 n-3 n-6 n-3 n-3 n-9 LBFDAU 3.3 ± 2.2 ± 2 ± 10.7 ±0.3 ± 0 ± 0.3 ± 2.9 ± 0.1 ± 1.6 ± 0.3 ± 0.3 ± (n = 16) 0.3 0.2 0.2 0.7 00 0 0.2 0 0.2 0.1 0 LBFDGG 2 ± 1.3 ± 1.9 ± 6.1 ± 0.3 ± 0 ± 0.3 ± 2.1 ±0.1 ± 1.1 ± 0.2 ± 0.1 ± (n = 36) 0.3 0.2 0.2 0.7 0 0 0 0.2 0 0.2 0.1 0LBFGKN 2.1 ± 1.2 ± 1.8 ± 6 ± 0.3 ± 0 ± 0.3 ± 2.1 ± 0.1 ± 1 ± 0.2 ± 0.2 ±(n = 36 + 60) 0.3 0.1 0.2 0.6 0 0 0.1 0.2 0 0.1 0 0 LBFIHE 2.1 ± 1.2 ±2.4 ± 6.7 ± 0.3 ± 0 ± 0.3 ± 1.9 ± 0.1 ± 1.2 ± 0.2 ± 0.2 ± (n = 36) 0.20.1 0.3 0.6 0 0 0.1 0.2 0 0.2 0.1 0 LBFLFK 3.3 ± 1.9 ± 1.9 ± 8.2 ± 0.3 ±0 ± 0.5 ± 3.2 ± 0.1 ± 1.4 ± 0.5 ± 0.3 ± (n = 36) 0.3 0.2 0.2 1 0 0 0 0.40 0.3 0.1 0.1 LBFPRA 2.3 ± 1.2 ± 3.8 ± 9.6 ± 0.3 ± 0 ± 0.3 ± 2.4 ± 0.1 ±1.1 ± 0.1 ± 0.2 ± (n = 36) 0.3 0.2 0.5 1 0 0 0 0.3 0 0.2 0 0.1

TABLE 4 Compositional analysis of T3 seeds of T2 plants cultivated inUSDA growth zones 3a-4b and 5a for field trials of canola eventscontaining the T-DNAs of plasmid VC-VC-LTM593-1qcz rc. The events areindicated in the first column. The analysis has been done on 4 BULK,whereby each BULK is a representative sample of all seeds harvedted from4 different geographic regions. Alpha-Tocopherol (mg/100 g seed), Beta-Tocopherol (mg/100 g seed), Delta-Tocopherol (mg/100 g seed),Gamma-Tocopherol (mg/100 g seed), Total Tocopherol (mg/100 g seed). Allresults have been normalized to the seed weight of seeds having 0%moisture. Values are the least square means. Means that are not sharinga letter are significantly different at the 95% confidence level. Alpha-Beta- Delta- Gamma- Tocopherols Event Tocopherol Tocopherol TocopherolTocopherol (VitE) Oil (%) LBFDAU 13.3 ab 0.25 a 0.58 a 29.5 a 43.7 a37.716 bcd LBFDGG 14.1 ab 0.23 a 0.45 bcd 25.6 b 40.4 abc 38.612 abcdLBFGKN 12.9 b 0.23 a 0.52 abc 26.9 ab 40.6 abc 39.400 abc LBFIHE 13.2 ab0.23 a 0.45 bcd 22.0 cd 35.9 cde 39.639 abc LBFLFK 12.5 b 0.23 a 0.52abc 25.7 b 38.9 abc 37.233 cd LBFPRA 13.6 ab 0.22 a 0.47 bcd 24.9 bc39.2 abc 39.189 abcd Topas 14.7 ab 0.25 a 0.36 d 16.6 e 31.9 e 36.581 dKumily 12.3 b 0.23 a 0.54 ab 24.4 bc 37.5 bcd 38.722 abcd Control 1*16.6 a 0.25 a 0.43 cd 24.1 bc 41.4 ab 38.923 abcd Control 2* 12.0 b 0.20a 0.45 bcd 20.8 d 33.5 de 40.567 a *Controls 1 and 2 are not Kumilybackgrounds

TABLE 5 Pearson correlation coefficients between fatty acids andtocopherols from T3 seeds of T2 plants cultivated in USDA growth zones3a-4b and 5a for field trials of canola events containing the T-DNAs ofplasmid VC- VC-LTM593-1qcz rc. Correlations that are significant areindicated with ***(p < 0.05) or with *(p < 0.10). Alpha- Beta- Gamma-Delta- Tocopherols Fatty Acid Tocopherol Tocopherol TocopherolTocopherol (VitE) ARA (20:4n-6) 0.059 −0.151 −0.162 −0.167 −0.129 EPA(20:5n-3) 0.030 0.102 0.372* 0.488*** 0.389* DPA (22:5n-3) 0.080 −0.0350.001 0.147 0.056 DHA (22:6n-3) 0.222 0.449*** 0.319 0.543*** 0.447***total n-3 (>20 C.) 0.029 0.159 0.416*** 0.566*** 0.432*** total n-6 (<20C.) −0.082 −0.119 0.143 0.218 0.096

1. A method for producing seed having increased tocopherol content,comprising: (1) providing a transgenic Brassica plant comprising atleast one recombinant polynucleotide encoding a delta-12-desaturase, atleast one recombinant polynucleotide encoding a delta-6-desaturase, atleast one recombinant polynucleotide encoding a delta-6-elongase, atleast one recombinant polynucleotide encoding a delta-5-desaturase, atleast one recombinant polynucleotide encoding a delta-5-elongase, atleast one polynucleotide encoding a delta-4-desaturase, and at least onerecombinant polynucleotide encoding an omega-3-desaturase; and (2)growing the plant to obtain oil-containing seeds, wherein the seeds haveincreased tocopherol content and increased n-3 very long chainpolyunsaturated fatty acid (VLC-PUFA) content compared to seeds from awild-type control Brassica plant; wherein the increased tocopherolcontent is the total tocopherol content and/or the content ofalpha-tocopherol; wherein the increased n-3 VLC-PUFA content comprisesEPA and/or DHA; and wherein the increased tocopherol content and theincreased n3 VLC-PUFA content in the seed of the transgenic Brassicaplant are positively correlated.
 2. The method of claim 1, wherein theincreased tocopherol content comprises the total tocopherol content, andwherein the total seed content of tocopherol is more than 35 mg/100 gseed.
 3. The method of claim 1, wherein the increased tocopherol contentcomprises the total tocopherol content, and wherein the total seedcontent of tocopherol is from about 36 mg/100 g seed to about 44 mg/100g seed.
 4. The method of claim 1, wherein the increased tocopherolcontent comprises the content of alpha-tocopherol, and wherein the seedcontent of alpha tocopherol is more than 12 mg/100 g seed.
 5. The methodof claim 1, wherein the amount of VLC-PUFAs in the seed is increased byat least 5%, at least 10%, at least 15%, at least 20%, or at least 30%by weight compared to seed from a wild-type Brassica control plant. 6.The method of claim 1, wherein the amount of VLC-PUFAs in the seed isincreased by 5% to 30% by weight compared to seed from a wild-typeBrassica control plant.
 7. The method of claim 1, wherein the Brassicaplant is a plant of the species Brassica napus, Brassica carinata,Brassica juncea, Brassica oleracea, Brassica nigra, or Brassica rapa. 8.The method of claim 1, further comprising the step of obtaining an oilfrom the seeds, said oil having an increased tocopherol content.
 9. Themethod of claim 1, wherein the tocopherol content is measured as thenumeric average of seed tocopherol content of at least 100 plants. 10.The method of claim 1, wherein the increased n-3 VLC-PUFA contentcomprises DHA and wherein the increased tocopherol content and theincreased DHA content in the seed are positively correlated.
 11. Themethod of claim 1, wherein the control plant is a corresponding wildtype plant of the same species and variety as the transgenic Brassicaplant.
 12. Seed of a transgenic Brassica plant, the seed comprisingtocopherol and n-3 very long chain polyunsaturated fatty acids(VLC-PUFAs), wherein the seed has increased tocopherol content andincreased n-3 VLC-PUFA content as compared to seed from a wildtypecontrol Brassica plant, wherein the increased tocopherol content is thetotal tocopherol content and/or the content of alpha-tocopherol; whereinthe increased n-3 VLC-PUFA content comprises EPA and/or DHA; and whereinthe increased tocopherol content and the increased n3 VLC-PUFA contentin the seed of the transgenic Brassica plant are positively correlated.13. The seed of claim 12, wherein the increased tocopherol contentcomprises the total tocopherol content, and wherein the total seedcontent of tocopherol is more than 35 mg/100 g seed.
 14. The seed ofclaim 12, wherein the increased tocopherol content comprises the totaltocopherol content, and wherein the total seed content of tocopherol isfrom about 36 mg/100 g seed to about 44 mg/100 g seed.
 15. The seed ofclaim 12, wherein the increased tocopherol content comprises the contentof alpha-tocopherol, and wherein the seed content of alpha tocopherol ismore than 12 mg/100 g seed.
 16. The seed of claim 12, wherein the amountof VLC-PUFAs in the seed is increased by at least 5%, at least 10%, atleast 15%, at least 20%, or at least 30% by weight compared to seed of awild-type Brassica control plant.
 17. The seed of claim 12, wherein theamount of VLC-PUFAs in the seed is increased by 5% to 30% by weightcompared to seed of a wild-type Brassica control plant.
 18. The seed ofclaim 12, wherein the seed is from a Brassica plant of the speciesBrassica napus, Brassica carinata, Brassica juncea, Brassica oleracea,Brassica nigra, or Brassica rapa.
 19. The seed of claim 12, whereinthere is no significant correlation between arachadonic acid (ARA)content in the seed and tocopherol content in the seed.
 20. The seed ofclaim 12, wherein the tocopherol content is measured as the numericaverage of seed tocopherol content of at least 100 plants.
 21. The seedof claim 12, wherein the increased n-3 VLC-PUFA content comprises DHAand wherein the increased tocopherol content and the increased DHAcontent in the seed are positively correlated.
 22. The seed of claim 12,wherein the control plant is a corresponding wild type plant of the samespecies and variety as the transgenic Brassica plant.
 23. A method forproducing seed having increased tocopherol content, comprising: (1)providing a transgenic Brassica plant comprising at least onerecombinant polynucleotide encoding a delta-12-desaturase, at least onerecombinant polynucleotide encoding a delta-6-desaturase, at least onerecombinant polynucleotide encoding a delta-6-elongase, at least onerecombinant polynucleotide encoding a delta-5-desaturase, at least onerecombinant polynucleotide encoding a delta-5-elongase, at least onepolynucleotide encoding a delta-4-desaturase, and at least onerecombinant polynucleotide encoding an omega-3-desaturase; and (2)growing the plant to obtain oil-containing seeds, wherein the seeds haveincreased tocopherol content and increased n-3 very long chainpolyunsaturated fatty acid (VLC-PUFA) content compared to seeds from awild-type control Brassica plant; wherein the increased tocopherolcontent is the total tocopherol content and/or the content ofalpha-tocopherol; wherein the increased n-3 VLC-PUFA content comprisesDHA; and wherein the increased amount of tocopherol and the increasedDHA content in the seed are positively correlated; wherein there is nosignificant correlation between arachadonic acid (ARA) content in theseed and tocopherol content in the seed; wherein the tocopherol contentis measured as the numeric average of seed tocopherol content of atleast 100 plants; and wherein the control plant is a corresponding wildtype plant of the same species and variety as the transgenic Brassicaplant.
 24. The method of claim 23, wherein the total seed content oftocopherol is more than 35 mg/100 g seed and/or wherein the seed contentof alpha tocopherol is more than 12 mg/100 g seed; and wherein theamount of VLC-PUFAs in the seed is increased by at least 5%, at least10%, at least 15%, at least 20%, or at least 30% by weight compared toseed from a wild-type Brassica control plant.
 25. The method of claim23, wherein the increased tocopherol content comprises the totaltocopherol content, and wherein the total seed content of tocopherol isfrom about 36 mg/100 g seed to about 44 mg/100 g seed.
 26. The method ofclaim 25, wherein the amount of VLC-PUFAs in the seed is increased by 5%to 30% by weight compared to seed from a wild-type Brassica controlplant.
 27. Seed of a transgenic Brassica plant, the seed comprisingtocopherol and n-3 very long chain polyunsaturated fatty acids(VLC-PUFAs), wherein the seed has increased tocopherol content andincreased n-3 VLC-PUFA content as compared to seed from a wildtypecontrol Brassica plant, wherein the increased tocopherol content is thetotal tocopherol content and/or the content of alpha-tocopherol; whereinthe increased n-3 VLC-PUFA content comprises DHA; and wherein theincreased amount of tocopherol and the increased DHA content in the seedare positively correlated; wherein there is no significant correlationbetween arachadonic acid (ARA) content in the seed and tocopherolcontent in the seed; wherein the tocopherol content is measured as thenumeric average of seed tocopherol content of at least 100 plants; andwherein the control plant is a corresponding wild type plant of the samespecies and variety as the transgenic Brassica plant.
 28. The seed ofclaim 27, wherein the total seed content of tocopherol is more than 35mg/100 g seed and/or wherein the seed content of alpha tocopherol ismore than 12 mg/100 g seed; and wherein the amount of VLC-PUFAs in theseed is increased by at least 5%, at least 10%, at least 15%, at least20%, or at least 30% by weight compared to seed from a wild-typeBrassica control plant.
 29. The seed of claim 27, wherein the increasedtocopherol content comprises the total tocopherol content, and whereinthe total seed content of tocopherol is from about 36 mg/100 g seed toabout 44 mg/100 g seed.
 30. The seed of claim 29, wherein the amount ofVLC-PUFAs in the seed is increased by 5% to 30% by weight compared toseed from a wild-type Brassica control plant.